Promoter Family Member (U037O11+D038O22)
Srini Devadas, David Gray, Ronny Krashinsky, Debra Lin, and Chris Zheng Liu
This part involves the -10 binding site, the -35 binding site, and the twenty nucleotides between that constitute the lux box. With this part, LuxR functions as a acyl-homoserine lactone-dependent repressor. LuxR resonds to the HSL produced by LuxI, N-(3-oxohexanoyl)-HSL. The Lux box is positioned such that it partially overlaps the consensus -35 and -10 hexamers of an RNA polymerase binding site.
This part is from the bacteriophage M13 genome, bp 3086-3132 in M13K07. It directs transcription of M13 gene VII (BBa_M13507, BBa_M13007). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
New England Biolabs M13K07. Sequenced in 2002 by L. Panganaban.
Anderson Promoter with lacI binding site
Promoters of the Anderson Promoter Collection with lacI binding site derived from [http://parts.igem.org/Part:BBa_R0010 R_0010] to make them [http://openwetware.org/wiki/IPTG IPTG] iducible.
We choosed lacI-binding-site of part BBa_R0010 because we assumed older parts would be more reliable. The part is shipped in backbone [http://parts.igem.org/Part:BBa_J61002 J61002] because the functionality is directly visible by adding IPTG. RFP ([http://parts.igem.org/Part:BBa_E1010 E1010]) should get expressed then.
Assambly of [http://parts.igem.org/Part:BBa_J23110 J23110] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Promoter Family Member (U097O11+D038O22)
Promoter Family Member with Hybrid Operator (D001O17)
unidentified/uncharacterized CinR dependent
pspoIIE promoter (spo0A J01004, positive)
promoter from b. subtilis, activated by spo0A
This promoter is designed to bind to C2003.
The placement of the operator site was designed to be near optimal for steric interference between C2003 and RNA polymerase.
This part was inadvertently made while trying to make BBa_R2111. It contains a single base pair mutation in the -10 site of BBa_R2111.
Anderson promoter J23118 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Promoter Family Member with Hybrid Operator (D001O61)
Anderson promoter J23103 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
optimized (TA) repeat constitutive promoter with 21 bp between -10 and -35 elements
This part is from the bacteriophage M13 genome, bp 786-835 in M13K07. It directs transcription of M13 gene V (BBa_M13505, BBa_M13005). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Generator of (CHE)Cholesterol esterase. Attached to placI, the part is inducible by IPTG and Lactose. It works better in Lactose.
Anderson promoter J23117 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Promoter of the copA copper efflux gene from Escherichia coli K12. This promoter is induced in the presence of copper ions, regulated by the activator protein CueR (CopR).
optimized (TA) repeat constitutive promoter with 15 bp between -10 and -35 elements
This cold shock promoter is only active at temperatures lower than 30 degree Celsius.
Promoter Family Member (U073O11+D062O22)
As per BBa_K180002, omitting the RBS and STOP biobrick parts (B0034 and B0015) for speed and ease of construction.
Assess if the RBS (B0034) sequence is needed prior to GFP (E0040) whilst still able to test function of part K180000.
Promoter Family Member (U049O11+D002O22)
Arabinose induced promoter. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Template for Building Primer Family Member
This promoter is repressed by tetR and lacI; it contains two binding sites for TetR and one LacI binding site. this part is an AND-gate since transcription occurs only in the presence of both ATC and IPTG.
Also designed by Pallavi Penumetcha and Robert Cool.
This part consists of the R0040 tetR promoter with the addition of the R0010 lacI binding site.
This is a promoter designed to be repressed by BBa_C2006: a MBP.Zif268.GCN4 homodimer.
The operator site is positioned to increase the steric hindrance between the repressor and RNAP based on comparing the structures of the repressor bound to DNA and RNAP bound to DNA.
This sequence is derived from BBa_R2000 and the protein structures of Zif268.GCN4(-2) bound to DNA and RNAP bound to DNA.
Promoter sequence for constituitive expression
There is a G->T point mutation of pSpac at the -1 site, changing the promoter to a stronger one - hence the name pSpac-hy, hy meaning hyper. This promoter drives transcription at rates of about ten times that of the wild type (pSpac) promoter.
Comes from the pPL82 vector, which is intended as a B. subtilis integrational vector. It is placed in front of the MCS in the plasmid, as it would drive the expression of the gene cloned into the MCS both in B. subtilis and E. coli.
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PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of rfp gene, which together is connected to vector PSB1C3 afterwards, and therefore we are able to observe the OmpR promoter efficiency through the control of light.
We constructed this biobrick through pcr.
Anderson promoter J23114 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the expression of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. This composite part does not have an LVA degradation tag at its end, hence can be used with all prokaryotic assemblies.
The part was designed and assembled from the iGEM2009 kit with standard RFC10 assembly.
The AraC promoter is found naturally in E.Coli at 1.52 min/centisome in the clockwise orientation. More information can be found at [http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene].
In native e.coli, this promoter is located upstream of FecA cds. Regulated by FecI protein, which is in turn regulated by FecA.
Promoter from the copper-sensetive CusR/CusS two component signal system. (E.Coli)
Sequence from the NCBI database, with added BioBricks prefix and suffix.
ftsAZ promoter is a promoter with levels of expression are inversely proportional to the growth rate. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0021/al0022 on MG1655 (804bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112320 ----------------------------------------------- al0021 Forward Biobricking of ftsAZ promoter CCATAGAATTCatgAGATCTaacgacagtgttggtgagcg al0022 Reverse Biobricking of ftsAZ promoter CGttaGGATCCgtaccaatctccagtcctactaccag
Promoter of flgA gene from E. coli flagella
''fliA'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGAGCATATCTCCTCCGCAGGTATCAAAAT *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAACAGTATCGCGATGATCGCCACGCTACGT
This part comes from the genome of ''E.coli'' K12 strain MG1655.
This is the PF promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PF promoter.
no special considerations seemed necessary
The original source is the P2 phage genome. For our work the DNA came from plasmids, kindly supplied to us by Prof. Calendar, University of California (Berkeley): Bacteriophage PSP3 and fR73 Activator Proteins: Analysis of Promoter Specificities; Julien and Calendar, 1996; JOURNAL OF BACTERIOLOGY, Oct. 1996, p. 5668???5675
Invertible sequence of dna includes Ptrc promoter
Seuqence is inverted by the FimE protein. Inside recognition sequence is a Ptrc promoter sequence. Switch is irreversible.
POPS regulated and Lac inverter system with strong RBS
The biobricks BBa_Q04121 and BBa_B0034 where used to build this biobrick
This part contains the LexAoperator and MajorLatePromoter genes with restriction site KdeI at the 5 prime end and AscI at the 3' end. Will use it as both a regulatory part for certain signals as well as a promoter.
Had to make sure the restriction sites used were not in the sequence or in the sequence of any of the genes downstream of the promoter.
This promoter normally represses the ColE2 activity gene, ceaB, in the ColE2 operon. To activate this promoter would require a RecA positive strain, as the protein LexA represses the promoter. To induce this promoter, an intercalating agent, mitomycin C, was utilized to induce the SOS response, and thus the expression of the genes under control of this promoter.
Lab strains of E. coli, such as Top10 from Invitrogen, are deficient in RecA so we required a RecA positive strain to induce this promoter. We used BL21 from Novagen.
This part comes from the ColE2 operon, which came from a plasmid.
This is the hybB promoter, which normally controlls hydrogenase regulation at low temperatures. It turns ON at 30oC and is active as low as 20oC (lower not tested). It is OFF at 37 and higher temperatures. In the literature, it is also active in anaerobic environments. This hasn't been tested.
lizjaneclarke@gmail.com Univerity of California - San Francisco
constitutive promoter with (TA)9 between -10 and -35 elements
With 9 TA repeats between the -10 and -35 elements, the distance between the two elements is not optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'off' state.
This part was PCR amplified from two synthesized primers that bound to each other.
This is the promoter sequence of g3 which has been separated from g8. It also includes the restriction enzyme EcoRV in front.
This part used in conjunction with BBa_M131520 allows g8 and promoter of g3 to be separate from each other, therefore allowing free regulation of g3 with no consequences on g8.
The p22 cII regulatory region sequence is a 97 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. p22 cII repressor protein, BBa_C0053, binds to it. This segment contains O-R1, O-R2, a fragment of O-R3, the -35 of P-RM, and P-R (-10 and -35 from Tom Knight)
AND-type promoter synergistically activated by cI and CRP
Luminesensor repressible recA408 promoter
recA promoter in 408 form.recA promoter belongs to the SOS regulon family, and promotes in E.coli transcription of recA protein, which forms filament when bound to ssDNA and trigger the self-cleavage of LexA protein.recA408 have one SOS box, which binds to the dimerized LexA protein and cause inhibition of the transcription of downstream gene.
The original RecA was mutated to 408 form in order to be responsive to 408 mutant form of LexA DNA binding domain.
LacI-repressible promoter A81 developed by Sidney Cox (http://www.nature.com/msb/journal/v3/n1/full/msb4100187.html). Showed a regulatory range (ON/OFF) of 1.5x10^4.
Pmom is one of the four late promoters of the Mu bacteriophage. It can be activated by the C protein, which is produced by the middle promoter Pm. Pmom works because of a special configuration of the DNA. It can be used to create a sensitivity tuner alongside the C protein of the same phage. This construction can be used to increase the POPS output of a promoter.
The design of this part had no special considerations
Synthetized from the reported sequence of Pmom promoter from Mu bacteriophage
FliA is a transcription factor involved in the formation of the flagella in E. coli. The promotor of fliA is induced by FlhDC. FliA seems to have a positive autoregulation on its promotor.
This part comes from the genome of E. coli K12 strain MG1655.
promoter of Paenibacillus sp. WLY78 nif gene cluster
The promoter of Paenibacillus sp. WLY78 nif gene cluster, We named it Pnif. Pnif is a σ70-dependent promoter located upstream of nifB. Pnif is in control of all the gene clusters of nitrogen fixation genome. Confirmation of nif promoter We standardized the nif promoter of Paenibacillus sp. WLY78, and linked it to the upstream of 3 reporter genes (RFP reporter: BBa_K1357010, YFP reporter: BBa_E0430;amilCP reporter: BBa_K1357009) to confirm the reliability of this promoter. The results are shown in Figure 2. A B C http://2015.igem.org/wiki/images/b/b2/Confirmation_of_Pnif_AB.png http://2015.igem.org/wiki/images/thumb/3/34/C-confirmation_of_Pnif.jpeg/622px-C-confirmation_of_Pnif.jpeg Fig.4. Confirmation of nif promoter of Paenibacillus sp. WLY78. (A) Pnif+RFP reporter; (B) Pnif+YFP reporter;(C)Pnif+amilCP reporter. The results confirmed the reliability of Pnif. The initiation of Pnif does not require inducement. Thus it is a effective constitutive promoter.
effeciency of the promoter in E.coli for ordinary parts
Pnif is from Paenibacillus sp. WLY78 genome,located upstream of nifB,the first gene of nitrogen fixation cluster.
Right facing promoter (for xylA) controlled by xylR and CRP-cAMP
removed RBS cut out several xylR activation sites
CRP-cAMP transcriptional dual regulator activates transcription
XylR-Xylose transcriptional activator activates transcription
Rock-paper-scissors - Paper primary plasmid
Composite part, consisting of BBa_K180005 and BBa_K180015, but with BBa_K180005 reading along the reverse strand, and BBa_K180015 reading along the forward strand. Forms the 'paper' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180005 reading along reverse strand, and BBa_K180015 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180005) has no effect over GFP or RhlI expression in BBa_K180015.
See relevant biobrick component parts (BBa_K180011 and BBa_K180012).
The pRE coding region from λ phage. It is able to be induced by CII (BBa_K116602).
THIS PART DOES NOT WORK. Need to add the CI operator sites.
---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Biobricking of cadC promoter and cadC gene PCR cadC-F and cadC-R on MG1655 (2336 bp, EcoR1/BamH1) Sub into pBca1256 (EcoR1/BamH1 910+2472bp, L) Product is pBca1256-K112406 ---------------------------- cb10013-cadC-F cgataGAATTCatgAGATCTcaccagcaatttgcgggcatatc cb10012-cad-R cgtagGGATCCtgaccagaccaaactggcg
MC31-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter It is a test of our positive feedback circuit for microcystin.
MC31, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
Promoter Family Member with Hybrid Operator (D001O27)
Promoter Family Member (U073O11+D014O22)
This promoter sequence is a derivative of part BBa_R0011 which is a -35 and -10 recognition site with two lac operator 1's inserted in the spacer region of the RNAP recognition region and before the -35 RANP recognition region. This part utilizes the UV5 double mutation at the -10 RNAP recognition site to recruit RNAP without the help of a CAP binding region.
The length of the fragment makes it cumbersome to gel purify
UV5 -10 RNAP recognition region double mutation
The -35 box of R0065 has been modified to promote constitutive (LuxR+3OC6HSL-independent) gene expression. An extended -10 sequence has also been added to increase absolute transcription. The promoter still contains the operators OR1 and OR2 such that it retains its sensitivity to CI.
Promoter Family Member (U097NUL+D014O22)
A Double Reporter (LacZ-alpha and RFP) loaded on pSB3C5. For more information please refer to BBa_I732402.
Rock-paper-scissors - Scissors primary plasmid
Composite part, consisting of BBa_K180018 and BBa_K180019, but with BBa_K180018 reading along the reverse strand, and BBa_K180019 reading along the forward strand. Forms the 'scissors' primary plasmid in Team:Southampton's rock-paper-scissors project design.
BBa_K180018 reading along reverse strand, and BBa_K180019 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180018) has no effect over mBanana or LuxI expression in BBa_K180019.
See relevant biobrick component parts (BBa_K180018 and BBa_K180019).
Anderson promoter J23106 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Promoter (luxR & HSL regulated -- lux pL)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr
The lux cassette of V. fischeri contains a left and a right promoter. The left promoter gives weak constitutive expression of downstream genes.This expression is down-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription from Pr, repressing transcription from Pl
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below.Includes most of Lux reulatory region, including the LuxR binding site which activates the right promoter. A putative LuxR autorepression binding site is also identified adjacent to the -10 site of the right promoter. This 2nd site has 55% identity with the first site. Putative inverted repeats (of size 18-27 bp) also exist between these two sites (not marked above), which may represent binding sites for other regulatory proteins. <img src="<bb_file>Image01.gif</bb_file>" width="614" height="362"> Unspecified.
Promoter, Zif23 regulated, test: between
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is between the -10 and -35 sites.
consensus sequences based on [Jensen98].
Game of Life - Secondary plasmid [tac pR, LuxI generator]
iGEM2009 Team:Southampton Game of Life secondary plasmid.
Regulated expression of LuxI (C0161) by TAC pR (K180000), a functional hybrid lac/trp promoter.
See relevant basic biobrick parts (K180000, B0034, C0161, B0015).
bolA promoter is a promoter where levels of expression are inversely proportional to the growth rate. To read more, go to following link [http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=402084&blobtype=pdf] This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR al0017/al0018 on MG1655 (467bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112318 ----------------------------------------------- al0017 Forward Biobricking of bolA promoter CCATAGAATTCatgAGATCTtgctgtggcagtgtaatcgtc al0018 Reverse Biobricking of bolA promoter CGttaGGATCCcttctatccgctcacgtatc
Pdps promoter. This part is actually in BBb format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR JH089F/JH089R on Salmonella L2. (379 bp, EcoRI/BamHI/DpnI) Sub into pBjh1601CK (EcoRI/BamHI, 3134+910, L) Product is pBjh1601CK-K112322 ----------------------------------------------- JH089F Forward biobricking of {Pdps} cgataGAATTCatgAGATCTaacgattcgacgcgaaccgc JH089R Reverse biobricking of {Pdps} cgttaGGATCCaatctcatatcctcttgatg
Vikram Vijayan, Allen Hsu, Lawrence Fomundam
Constitutive Promoter, Medium Transcription
LacI-repressible promoter B4 developed by Sidney Cox (http://www.nature.com/msb/journal/v3/n1/full/msb4100187.html). Showed a regulatory range (ON/OFF) of 2.5x10^4.
Double Promoter (cI, negative / LacI, negative)
Promoter Family Member (U097NUL+D038O22)
K091146 is a modified version of the pLas' promoter (BBa_K091117) containing the addition of a luxR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of PAI-1 and LasR and repressed in the presence of AI-1 and LuxR.
The Lux box was placed adjacent to the LasR binding site with the aim of minimizing cross-talk in the presence of both PAI-1 and AI-1; since the LasR site should have a higher affinity, we hope that the binding of PAI-1 will competitively exclude the binding of AI-1.
The LuxR binding site is from BBa_R0062 and the pLas' promoter is from the region upstream of the LasI gene in Pseudomonas aeruginosa.
This is the promoter for the ''Eschaerichia coli'' JM109 ''cstA'' gene. It includes the CRP-binding site and the RNA polymerase-binding site. Low glucose concentration results in increased activity by adenylate cyclase. cAMP binds to the cAMP receptor protein, which, in its bound form, is able to associate with the promoter and promote transcription of the downstream gene. (''cstA'' encodes the carbon starvation protein.)
This hybrid promoter is designed to respond to activation by LuxR when it is bound to AHL and to repression by the phage lambda cI repressor. The repression needs to win over activation.
This part is based on the idea of replacing the region of the Lux promoter downstream of the -35 with the OR2 cI half-operator, added LuxR-10 region. Based on what we have found up to now, promoters which are constructed just by inserting OR1/2 downstream of Lux are all very leaky.
this part comes from LuxR promoter and cI lambda promoter.
luxR operator (lux box) was deleted from R0065 and replaced with a BamHI hexamer.
added a bamHI site immediately upstream of -35 in place of lux operator
constitutive promoter which outputs .3 TIPs point mutants of wild type rrnB promoter P1
Promoter Family Member (U037O11+D050O22)
The MioC promoter is a cell-cycle dependent promoter that is repressed before initiation of replication and depressed shortly after. The regulation of the MioC gene is overseen by several DNAa proteins, which bind along the promoter. This particular MioC promoter was isolated from K-12 (JM109). This promoter can be used in series with a reporter to signal only during a given part of the cell cycle.
Site directed mutagenesis was used to create a Spe1 restriction site.
The source of this part is from the genomic dna of E.coli (JM109).
PphoA promoter is a phosphate-responsive promoter. In high concentration of phosphate, it will be repressed while in low phosphate concentration, it will be activated.
Operator for RcnR(YohL) the negative regulator of niquel extrusion pump(RcnA) in E.coli
We needed the RcnR operator without its promoter, so it was necessary to predict the promoter and discard it from the sequence.
Promoter Family Member (U085O11+D062NUL)
Promoter Family Member (U085O11+D026O22)
This promoter is a modified version of the Mnt promoter that is also responsive to TetR. The promoter should be repressed by Mnt repressor. It should also be repressed by TetR, and in the absence of Mnt repressor, should be induced by aTc.
Mnt repressor binds as a tetramer to two half-operator sites. Introduction of TetR binding sites between the Mnt promoter -10 sequence and the start site for transcription should allow for repression by TetR. Accordingly, the hybrid promoter was designed by 1) Remove all bases of mnt promoter 3??? to -10: gagtcgtattaattt will be replaced by tccctatcagtgata, and 2) truncate the second half of ptet -10 and mutate -35 so that it function to bind tetR (replace ttgaca with actgta), but is not a promoter, and 3) place the tetR1 and tetR2 binding sites 3??? to truncated mnt -10 sequence.
This part comes from the Mnt promoter and the TetR promoter
This part is from the bacteriophage M13 genome. It directs transcription of M13 gene II (BBa_M13502, BBa_M13002). Promoter identified in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Pm is the middle promoter of the Mu bacteriophage. It is in charge of the transcription of the activator protein C. Pm is activated by a product of the early transcription, protein Mor. It can be used to build a sensitivity tuner alongside this protein, this construct can be used to increase the amount of POPS in a certain genetic construction.
Synthetized from the reported sequence of Pm promoter from Mu bacteriophage.
Assambly of [http://parts.igem.org/Part:BBa_J23106 J23106] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is after the -10 site.
Dual-Repressed Promoter for p22 mnt and TetR
This part is a promoter that is repressed by both p22 mnt and TetR. It can be used to control the response of any gene that follows this promoter. It will be repressed by the presence of either p22 mnt or TetR.
TetR usually binds to two operator sites, one located upstream from -35 and one located between -35 and -10. However, p22 mnt also has two operator sites, one between -35 and -10 and another downstream from -10. Since each had an operator site in between -35 and -10, we had to eliminate the TetR operator site in order to allow the promoter to work. TetR was picked because it has a stronger binding than p22 mnt, and so would be more able to use only one site to bind.
This part was designed by combining sequences from parts C0040 (TetR promoter) and C0072 (p22 mnt promoter).
cI repressor from phage TP901 infecting L. lactis
position 5 mutated promoter lux pR-5 (luxR & HSL regulated)
The position 5 mutated promoter lux pR-5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-5 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-5 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
By PCR method the G residue of position 5 was changed to a C based on the sequence of the promoter lux pR(BBa_R0062).
Promoter Family Member (U097NUL+D062O22)
The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207] although they should be same. We choosed part BBa_R0010 because we assumed older parts would be reliable.
Assambly of [http://parts.igem.org/Part:BBa_J23100 J23100] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010]
This is the Psid promoter taken from the P4 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the Psid promoter.
This part is from the bacteriophage M13 genome, bp 381-428 in M13K07. It directs transcription of M13 gene X (BBa_M13510, BBa_M13010). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the Lux repressor, LuxR. Two molecules of LuxR protein form a complex with two molecules the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.In thisversion, a binding site for cI dimer has been added: binding to this site dominantly represses transcription.
V. fishceri and phage lambda (courtesy of Ron Weiss)
This biobrick has a promoter inducible by PAI+LasR (BBa_R0079)and produces LuxI enzyme (BBa_F1610). This enzyme produces 3OC6HSL (AI).
The DNA of BBa_R0079 and BBa_F1610 biobricks where used.
Promoter Family Member (U073O11+D002O22)
This is a LuxR-30C6HSL-independent version of R0065. All DNA upstream of the -10 box is from the constitutive promoter J23119. All DNA downstream from the -10 box down is from R0065. This results in the replacement of the Lux box region for a constitutive promoter region and the maintenance of the two cI operators downstream of the transcription start site.
Promoter Family Member (U049O11+D014O22)
R0051 was modified to contain consensus -35 and -10 sequences. The CI operator OR1 was moved from it's original position (overlapping the -10) to between the -35 and -10. Fortuitously this results in the optimal (for high expression) 17bp spacing between the -35 and -10 sites.
Promoter (lacI regulated, lambda pL hybrid)
Neelaksh Varshney, Grace Kenney, Daniel Shen, Samantha Sutton
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The PLlac 0-1 promoter is a hybrid regulatory region consisting of the promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. The hybrid design allows for strong promotion that can nevertheless be tightly repressed by LacI, the Lac inhibitor (i.e. repressor) (<bb_part>BBa_C0010</bb_part>) ([LUTZ97]). The activity of the promoter can be regulated over a >600-fold range by IPTG in E.Coli DH5-alpha-Z1 (same paper reference).
hybrid promoter design to create strong promoter that is, at the same time, highly repressible. note that the upstream operator installed in this hybrid is slightly different than the one in the original source (Lutz and Bujard, 1997). the most upstream operator region is slightly truncated in the represillator version, so that both operators in the hybrid are the same sequence. see references for details. also, the sequence has been truncated after the transcriptional start site. LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and increase transcription. This part is incompatible with environments containing lactose or lactose analogs.
represillator of Elowitz and Leibler (2000)
Promoter (lambda cI and luxR regulated -- hybrid)
cI repressor negatively regulates this promoter and LuxR activates its transcription.The effect of cI is dominant over LuxR. This part is based on the LuxR and cI repressor regulated hybrid promoter designed and tested by Ron Weiss. It requires the binding of two cI repressor dimers for maximal repression and contains two cI repressor binding sites namely, OR1 and OR2. This promoter is leaky in the sense that 'some' transcription is seen in the absence of both cI and LuxR. LuxI cI activity of promoter + + zero + - maximum - + zero - - leaky (no quantitative information)
This part was designed based on the LuxR and cI repressor regulated hybrid promoter tested by Ron Weiss and the LuxR-LuxICDABE sequence annotated by Tom Knight AF170104 .
PenP operator from Bacillus Lichenformis. Two operator sites are negatively regulated by PenI (C0074).
This is a pRecA promoter with LexA binding sites. LexA is cleaved by RecA when RecA is upregulated due to induction of the SOS response triggered by single stranded DNA. For this part to work it must be in a RecA+ strain of E. coli such as TG1
How far past the start site of transcription do you need to go in order for the promoter to drive transcription? The promoter has been confirmed to drive transcription in a regular DH5alpha strain, so it the answer to the question is, 3 bp before the suffix, maybe less. The LexA binding site is in the core region of the promoter (between the -35 and -10 sigma 70 binding sites). According to Robert Sidney Cox III and colleagues in their paper "Programming ene expression with combinatorial promoters, the core region is the most effective region for binding of repressor molecules.
synthetic. Oligonucleotides were designed to form the double stranded molecule with sticky ends for direct ligation into a biobrick vector.
Promoter Family Member (U049O11+D038O22)
The Rec A(SOS) promoter has been PCR amplified from the chromosomal DNA from MG1655(Ecoli).
optimized (TA) repeat constitutive promoter with 17 bp between -10 and -35 elements
Binding region for LasR protein (positive regulation)
"Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Lynn Rust, Everett Pesci, and Barbara Iglewski
I0500 with extra trailing restriction sites: Eco-Xba-I0500-HindIII-NcoI-Spe-Pst
optimized (TA) repeat constitutive promoter with 13 bp between -10 and -35 elements
This is the PO promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PO promoter.
The source of the DNA is the P2 phage genome.
It is the same as BBa_I0050. But the part seems not working so we make a new one, which the template is from iHKU team.
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. This part also exist together with lacI, part BBa_K180000 [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Oligos ordered from SigmaAldrich were annealed to form a double stranded DNA segment, ready to be ligated into any BioBrick backbone.
Created from oligos ordered from SigmaAldrich. Oligo sequences: Ptac+ AATTCGCGGCCGCTTCTAGAGGAGCTGTTGACAATTAATCATCGGCTCGTATAATGTGTGGAATTGTGAGCGGATAACAATTTA Ptac- CTAGTAAATTGTTATCCGCTCACAATTCCACACATTATACGAGCCGATGATTAATTGTCAACAGCTCCTCTAGAAGCGGCCGCG
This is a fusion protein with Ag43, a protein from E coli tagged with a 6xHis tag on the N-terminal and sfGFP tagged on C-terminal of the pro-protein. Ag43 causes cell-cell adhesion in E coli, and the 6xHis and sfGFP tags are present to allow convenient study dependence of Ag43 mediated aggregation on Ag43 expression levels, which can be determined from the sfGFP fluoresence and/or quantification of 6xHis tag. The 6xHis tag is included to allow protein concentration estimation in case the sfGFP fails to fold properly.
The location for the 6xHis tag is the same as that for BBa_K2168000(Ag43 tagged with 6xHis on N-terminal under pBAD/araC promoter) and uses the same design principles. For independent folding, a flexible GGGGSGGGGS linker was added between Ag43 and sfGFP, and the linker was choosen to be not very hydrophilic or hydrophobic, since Ag43 lies very close to lipid membranes.
This part was made from Gibson assemblies from fragments of two previous parts - BBa_K1352000 and BBa_I746908, which code for Ag43 and sfGFP respectively.
Promoter, Zif23 regulated, test: between and after
Promoter with standard -10 and -35 sites. A symmetric Zif23 dimer target site is between the -10 and -35 sites, and a symmetric Zif23 dimer target site is after the -10 site.
to be used with transcription factor BBa_J34802
NOT Gate Promoter Family Member (D001O33)
Promoter Family Member (U097O11+D050O22)
Hybrid promoter (trp & lac regulated -- tac pR)
The Ptac promoter is a functional hybrid promoter, derived from the trp and lac promoters, that are regulated by trp and lac [1]. The Ptac sequence has been extracted by PCR from the commercially available plasmid pMAL-pIII from New England Biolabs (N8101S), with the addition of the biobrick prefix and suffix sequences. [1] Proc. Natl. Acad. Sci. USA, Vol. 80, pp. 21-25
Sequence includes lacIq and Shine-Dalgarno sequence from pMAL-pIII prior to the Ptac promoter to ensure correct functionality of the promoter.
NikR promoter, a protein of the ribbon helix-helix family of trancription factors that repress expre
Nickel responsive transcriptional regulator
Promoter Family Member (U085O11+D038O22)
nhaA promoter, that can be regulated by pH and nhaR protein.
It's E.coli K12 sodium/proton anti-transport promoter. It can be a pH sensor. We test pH senses in different pH value and Sodium concentration.
This part is from the bacteriophage M13 genome, bp 1500-1547 in M13K07. It directs transcription of M13 gene III (BBa_M13503, BBa_M13003). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
NOT Gate Promoter Family Member (D001O77)
Promoter, Standard (HK022 cI regulated)
[Note: This is the same part as R0050 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The pR regulatory region is a 98 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two operator sites at which HK022 cI protein can bind cooperatively. The -35 and -10 regions from pR are included along with some flanking sequences. The cutoff for the 5' end of the regulatory region was placed just after the pM -10 region which promotes transcription of a gene upstream (thus the pM -10 region is not included). The cutoff for the 3' end of the regulatory region was placed immediately prior to the RBS.
Derived from STHK022N . Modified to comply with BBa_S0001: TTGACA-17N-GATACT Literature claims that there is no cross-talk between HK022 cI regulatory region, Lambda cI, 434 cI and P22 cI, but this has not been verified experimentally by the synthetic biology group at MIT.
Promoter for recA gene - SOS and Ultrasound Sensitive
According to Vollmer, et. al in their 1998 Applied and Enviormental Microbiology paper, "Bacterial Stress Responses to 1-Megahertz Pulsed Ultrasound in the Presence of Microbubble", the promoter for the recA gene, a known SOS response gene, is upregulated 4 fold when cells are exposed to high frequency ultrasound and microbubble cavitation.
We did not see the desired response from ultrasound exposure that we expected from this promoter. However, we did not induce the same acoustic cavitation that Vollmer et. al. used to elicit a response from this promoter.
Our microarray experiment showed that this gene was responsive to sound and ultrasound. Therefore, we cloned the promoter region located approximately 250 base pairs before the start codon for the cadB gene.
There was a relatively high basal level of expression of this gene that made this part difficult to incorporate into our lysis device design.
cloned from E. coli MG1655 genome located upstream of CadA and CadB genes
Promoter Family Member (U073O11+D062NUL)
Hybrid promoter constisting of two LacI O2 sites at the ends of the promoter and two recognition sites (OR2 and OR1) for the P22 c2 repressor. The promoter should function as a NOR gate.
a small fragment of OR3 remains.
LasR and 3OC12HSL regulated promoter with a single Tet Operator (binding site for TetR repressor)
pBAD is an E.coli promoter that is induced by L-arabinose. K206000 is a mutagenized pBAD promoter that is responsive to lower concentrations of arabinose than wild type ( I13453 ) and, additionally, exhibits a higher maximum of protein expression as measured by coupling to a fluorescent reporter.
There were no special design considerations.
The sequence was obtained by applying all mutations that upregulated AraC binding and subsequent promoter activity listed in reference [1].
AraC operator, truncated to include araO1, araI1, araI2, c-amp1, and c-amp2 sites. This operator should *activate* transcription in the presence of AraC; b/c the operator lacks the araO2 site, there should not be araC-mediated repression.
Edited Xylose Regulated Bi-Directional Operator 2
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has a scrambled CRP binding site and and altered RNA ploymerase binding site that was designed to more resemble the consensus sequence at an intermediate level.
Both directions were kept in the sequence of the part even though it is intended to work in the right facing direction because of possible looping that may occur as a transcriptional control. The RNA polymerase binding site was altered to make binding stronger, attempting to allow activation when XylR-xylose alone is bound (the absence of CRP-cAMP).
Taken from E. Coli genome bases 3728830-3729092, base pairs changed at RNA polymerase bining sites.
Paper - Primary plasmid (part 2) [tac pR, GFP and RhlI generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'rock' primary plasmid.
Regulated expression of RhlI (C0170) and GFP (E0040) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, E0040, B0015).
RHLR/RHLI Inducible & LasR/LasI repressible Promoter
This part is similar to BBa_R0079 but has an added binding site for RHLR/RHLI in between the -35 and -10 region. Thus this promoter is induced by LasR/LasI but repressed by RHLR/RHLi binding.
There is a 17bp spacer between the -35 and -10 regions and the binding site for RHLR/RHLI is 20bp long. I was able to fit the whole binding site in the spacer region because the last three base pairs of the -35 region and the first three base pairs of the RHLR/RHLI binding site are the same.
Rust L. et. al., "Analysis of the Pseudomonas aeruginosa Elastase (lasB) Regulatory Region". Journal of Bacteriology, 1996.
Promoter Family Member (U085O11+D014O22)
Assambly of [http://parts.igem.org/Part:BBa_J23103 J23103] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Modified lamdba Prm promoter (repressed by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI)
The O-R1 region of 434 contained 14 base pairs as opposed to the 17 base pairs of the O-R3 site of lambda. Also, it was noticed that the O-R3 site of the lambda included part of the -10 site. Hence, to preserve the spacing and the -10 site, the three nucleotides that were in both the -10 site and the lambda O-R3 site were retained. The 14 nucleotides that were in the O-R3 site and not in the -10 site were replaced with the O-R1 site of the 434.
Lead Promoter (Part 4) messed up EcoRI site
Game of Life - Primary plasmid (part 2) [lux pR, GFP and LacI generator]
Part of the iGEM2009 Team:Southampton Game of Life primary plasmid.
Regulated expression of LacI (C0012) and GFP (E0040) by PLUX promoter (R0062), controlled by presence/absence of AHL-LuxR.
See relevant basic biobrick parts (R0062, C0012, B0034, K180001, B0015)
This is a modification of the standard plac operon with everything before the -35 site removed, the Or2 site from the lambda promoter inserted at the beginning, and a 17-bp spacer added between the two.
Promoter Family Member (U073O27+D062NUL)
A part used to quantify the AraC-RBS construct K259007 created by the BCCS-Bristol iGEM'09 team.
This part was designed with the specified biobricks from the parts registry. AraC occurs naturally in E.coli. GFP occurs in jellyfish species, RBS designed by the Anderson family and Terminator by Resma Shetty.
this is a modified version of the LacIQ promoter that should bind LacIQ tighter than the original.
This is a modified version of the LacIQ promoter.
This is the PLL promoter taken from the P4 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PLL promoter.
This part is a heat shock promoter, it refers to the transcription factor sigma32. when the temperature rise, the transcription factor sigma32 expresses a lot and binds to the RNA ploymerase to recognize the conserved sequence in -35 and -10 region, then the promoter starts to transcript.
The whole sequence of this part is only 90bp, so take a futher consideration if you intend to digest it for use.
We align the HSP promoters from the literature and then find the conserved sequence, then we just modified the promoter of groES of E.coli, and synthesis the part through BIC genescript.
Inverted tetR promoter with noncoding, spacer DNA upstream of it to make the total length 650 bp.
This part is in a series of inverted tetR promoters that have total lengths of 150, 250, 350, 450, 650, and 850 bp. We chose the region upstream of tetR in part Q04400 to be the noncoding DNA segment. This noncoding segment consists of the 3' end of tetR and B0015. None of the parts in this series was long enough to include the start codon of tetR, so a functional tetR protein should not be transcribed.
Primers were synthesized to bind to part Q04400, and then a PCR reaction was run.
Dual-Repressed Promoter for p22 cII and TetR
This part was designed by combining sequences from parts R0040 (TetR promoter) and R0053 (p22 cII promoter).
Anderson promoter J23113 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Inverted tetR promoter with noncoding, spacer DNA upstream of it to make the total length 350 bp.
The promoter region (inclusive of regulator binding sites) of the B. subtilis RocDEF operon
This is the reported promoter region from the Bacillus subtilis RocDEF operon as taken from the DBTBS website (http://dbtbs.hgc.jp/). Base 1 to base 21 contains the binding site for the binding factor, RocR. The sequence, TATGCAAAAGAATTTTGCACT, reportedly contains the consensus sequence for binding of this factor. Base 42 to base 62 contains another binding site for the RocR binding factor, with ATATCAGAATGTTTTTGCACC as the binding consensus for this sequence. Bases 113-135 outline the binding site of the binding factor AhrC (CTTGCATTTATATAAAGGGAAAG). Bases 96 to 135 outline the promoter region for this operon (CTTGATTTGGCACAGAACTTGCATTTATATAAAGGGAAAG).
This sequence only outlines the reported promoter region as ascertained from the online resource described above, and may lack nucleotides not described by the operon analysis used.
Information was obtained from: http://dbtbs.hgc.jp/COG/prom/rocDEF.html.
constitutive promoter with (C)10 between -10 and -35 elements
With 10 C repeats between the -10 and -35 elements, the distance between the two elements is optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'on' state.
Promoter Family Member with Hybrid Operator (D001O21)
BBa_J23100 constitutive promoter directs the expression of tetracycline repressor (TetR). TetR binds to pTet regulatory region resulting in a ''negative control'' of the production of LuxR. The whole system acts as an ''Inverter'' of Tet system controlling LuxR expression. TetR repression is inhibited by the addition of tetracycline or its analog, [http://openwetware.org/wiki/ATc aTc].
The biobricks BBa_K145201 and BBa_K116640 where used for this biobrick
Assambly of [http://parts.igem.org/Part:BBa_J23116 J23116] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Promoter Family Member (U061O11+D026O22)
Promoter of fliL gene from E. coli flagella
''fliL'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon, ). Its promoter is consitutively repressed. It is activated by flhD4C2 and by fliA with different forces ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). A deeper characterization is in project
The primers used to amplify this sequence are : Forward : TCGAATTCGCGGCCGCTTCTAGAGCAAGGGCGTGTAACAGGCAAC Reverse : TCCTGCAGCGGCCGCTACTAGTAGTCATGTGTTGCGGTCTTCCTGTG
Rock-paper-scissors - Rock secondary plasmid [rhl pR, LacI generator]
Regulated expression of LacI (C0012) by Rhl pR (R0071).
See relevant basic biobrick parts (R0071, B0034, C0012, B0015)
Promoter Family Member (U097O11+D014O22)
Double Promoter (TetR, negative / P22 cII, negative)
sense TWO INPUTS, activation by AHL and repression by LacI.
Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
Promoter Family Member (U073O26+D062NUL)
Binds Maltose and stimulates transcription
tetR promoter with noncoding, spacer DNA upstream of it to make the total length 150 bp
This part is in a series of tetR promoter that have total lengths of 150, 250, 350, 450, 650, and 850 bp. We chose the region upstream of tetR in part Q04400 to be the noncoding DNA segment. This noncoding segment consists of the 3' end of tetR and B0015. None of the parts in this series was long enough to include the start codon of tetR, so a functional tetR protein should not be transcribed.
Modified lambda Prm promoter (OR-3 obliterated)
Lambda Prm promoter modified to be activated but not repressed by the lambda repressor (cI)
In wild type lambda phage, the OR3 site (-27 to -11) reads "tatcccttgcggtgata" on the sense strand of DNA. To prevent lambda repressor (cI) from binding to this site, the 4th through 10th nt of OR3 were replaced by the 7 nt between OR2 and OR1, "aaatagt" (-57 to -51), which in effect mutates 5 nt of the promoter. These nt were chosen to be about halfway between the -10 and -35 boxes of the promoter. Furthermore, since these nt already act as a spacer in wild-type phage, it is hoped they will not create undesired interactions.
NOT Gate Promoter Family Member (D001O55)
MC7-rbs-luxI-Ter-Pcons2-rbs-luxR-Ter-Prlux-rbs-OMPA-golB-rbs-luxI-Ter. It is a test of our positive feedback circuit for microcystin.
MC7, luxI, Pcons2, luxR, Prlux, OMPA-golB, Ter
LacZ constitutive promoter, without including operators.
Constitutive weak promoter of lacZ which sequence was changed at position 29 (T->G) in order to make it weaker
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
Sequence for pTet inverting regulator driven by the TetR protein.
BBa_R0040 TetR-Regulated Promoter is based on a cI promoter. It has been modified to include two TetR binding sites and the BioBrick standard assembly head and tail restriction sites.
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
Plac-rbs-rfp-Ter It produce rfp when there is enough IPTG or less lacI
Natural Xylose Regulated Bi-Directional Operator
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has an unscrambled CRP binding site and and unaltered promoter region (unlike BBa_K100001 through BBa_K100003).
Both directions were kept in the sequence of the part even though it is intended to work in the right facing direction because of possible looping that may occur as a transcriptional control.
Taken from E. Coli genome bases 3728830-3729092
Prlux-rbs-rfp-Ter. It produces rfp when there is enough luxR and AHL.
Promoter Family Member (U049O11+D050O22)
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes the three OmpR binding sites: C1, C2, and C3. The promoter starts at -107 and goes up to the transcriptional start site at +1. An alternate version, BBa_R0083, cuts out the C2 and C3 sites. The efficacy of this promoter versus the truncated ompC upstream region (BBa_R0083) or the ompF upstream region (BBa_R0084) is currently unknown.
Promoter Family Member (U061O11+D002O22)
recA408 Promoter + B0030 + GFP + ssrA-tag
This is an exact 'upside down' version of BBa_R0051. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. By adding this part, we wish to emphasise the possibility of engineering both DNA strands, and the additional regulatory potential that it gives.
Any 'upside down' (reverse complementary) sequences made by PCR amplification need be made with suffix and prefix is opposite order with respect to the original default sequence.
This is a modified version of the LacI promoter that should bind LacI tighter than the original.
This promoter is a modified version of the LacI promoter
Based on the AHL regulated BioBrick part R0062 and the previous work on LacI regulated promoter (ref. 1), we designed a new promoter regulated by AHL and LacI. Since this promoter is a kind of a "HYBRID" of the two, we call it HYBRID PROMOTER. Compared with existing BioBrick hybrid promoter R0065, this promoter is different in that its sequence is much shorter with lux box and -35 region overlapping, and that it is repressed by LacI, not cI. Also, the output is clearly detected as shown in Fig. 3.
Promoter Family Member (U061O11+D062O22)
TetR - TetR-4C heterodimer promoter (negative)
This promoter is repressed by a heterodimer of wild-type TetR and mutant TetR-4C. It differs from wild-type TetR promoter in that the +4 and -4 regions of its O2 binding site have been changed, and the -35 region of the reverse promoter (pA) has been destroyed.
The O2 and O1 mutations are all single point mutations, occuring at the first base which appears listed here.
Promoter Family Member (U049O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732402.
The pLux' promoter is a modified version of BBa_R0062 containing the -35 region TTTAAT in place of TTTACG. Transcriptional activation is expected to occur in the presence of LuxR and AI-1, just as with R0062. The modified -35 region was designed to serve as a control for the evaluation of BBa_K091156, the pLux+ Las- promoter.
This promoter is identical to pLux+ Las- with the exception of the Las box's absence
Promoter Family Member (U085O11+D050O22)
Taken from no other than Curious George, this promoter is induced by the presence of banana in the metabolism of any mammal. It is very weak however without the presence of the HngR protein. For transcription to reach significant levels both HngR and banana must be present
Monkeys can be dangerous when aggrevated
Composite part, consisting of BBa_K180005 and BBa_K180006, but with BBa_K180005 reading along reverse strand, and BBa_K180006 reading along the forward strand.
BBa_K180005 reading along reverse strand, and BBa_K180006 reading along the forward strand to ensure the constitutive promoter BBa_J23119 (present in BBa_K180005) has no effect over LacI or GFP-LVA expression in BBa_K180006.
See relevant biobrick component parts (BBa_K180005 and BBa_K180006)
The BBa_J23100 constitutive promoter with Lac systems inverter and strong RBS
The biobricks BBa_K266008 and BBa_B0034 where used to build this part
This part is from the bacteriophage M13 genome, bp 1155-1201 in M13K07. It directs transcription of M13 gene VIII (BBa_M13508, BBa_M13008). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. B0030 is a strong RBS based on Ron Weiss thesis
Placement of the operator sites was optimized for steric interference between the C2003 and RNA polymerase based on structural models.
The 434 cI regulatory region sequence is a 89 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. 434 cI repressor protein, <bb_part>BBa_C0052<bb_part>, binds to it. This segment contains O-R1, O-R2, P-R, and P-RM -35.
optimized (TA) repeat constitutive promoter with 19 bp between -10 and -35 elements
PompR is a OmpR-controlled promoter which can be positively regulated by phosphorylated OmpR. This promoter is taken from the upstream region of ompC. Phosphorylated OmpR binds to the three operator sites and activates transcription. We insert PompR(BBa_R0082) sequences into the upstream region of our target gene gp35 through restriction-ligation method, which together is connected to vector PSB1C3 afterwards, and therefore we are able to regulate the expression of in through the control of light. Bxb1 gp35 is a serine integrase in Mycobacterium phage Bxb1. This intergrase can exclusively catalyze site-specific recombination. The dismiss of light singal can drive the expression of integrase, which alone can set a DNA sequence(there it means the promoter) flanked by oppositional attB and attP sites, thereby produce an inverted sequence flanked by attL and attR sites. As a consequence, the promotor moves on to express the toxic protein instead of the attractant. We insert the gp35 gene into the downstream region of the PompR promotor by means of 3A synthesis so that the expression of integrase can be regulated by light. In addition, we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation. Meanwhile, we insert the recombined sites of attB and attP into the flanking sequences of our constitutive promotor (BBa_J23110 ) and so far we accomplish our reverse promotor system.
we add a 6N promotor between PompR and gp35 to decrease basal integrase expression above flipping threshold level which could lower the efficiency of int translation.
Anderson promoter J23100 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Modified lambda P(RM) promoter: -10 region from P(L) and cooperatively repressed by 434 cI
Lamdba P(RM) promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operator sites are used for both activation and repression to enhance cooperativity. Additionally, the -10 region from lambda P(L) was used instead of that from P(RM).
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage. This part differs from BBa_I12036 because the -10 region from P(L) was used instead of that from P(RM).
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40. ; Lutz, R & Bujard, H. "Independent and tight regulation of transcriptional units in Escherichia coli via the lacR/O, the TetR/O and AraC/I1-I2 regulatory elements, Nucleic Acids Research, 1997, Vol.25, No.6 (1203-1210)
GoL - Primary plasmid (part 1)/RPS - Paper primary plasmid (part 1) [LuxR generator]
Constitutive promoter selected to allow for continuous expression of C0062.
Modified lamdba Prm promoter (cooperative repression by 434 cI)
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operators are used for both activation and repression to enhance cooperativity.
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage.
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40.
promoter for FabA gene - Membrane Damage and Ultrasound Senstitive
According to Vollmer, et. al in their 1998 Applied and Enviormental Microbiology paper, "Bacterial Stress Responses to 1-Megahertz Pulsed Ultrasound in the Presence of Microbubble", the promoter for the fabA gene, a known membrane damage response gene, is upregulated seven fold when cells are exposed to high frequency ultrasound and microbubble cavitation.
optimized (A) repeat constitutive promoter with 18 bp between -10 and -35 elements
improved for transcription factor BBa_J34807 binding
Edited Xylose Regulated Bi-Directional Operator 1
This part contains the operator region controlling transcription for the xyl operon. This regulatory region is dependent upon the binding of CRP-cAMP and XylR bound to xylose for activation. Upon binding, transcription is activated in both directions (xylFGH and xylAB facing). This part has a scrambled CRP binding site and and altered RNA ploymerase binding site that was designed to more resemble the consensus sequence slightly.
araC-Pbad - Arabinose inducible regulatory promoter/repressor unit
Valentina Herbring, Sebastian Palluk, Andreas Schmidt
This promoter can be induced by arabinose. In presens of arabinose the promotor is active.
Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J Bacteriol. 1995 Jul;177(14):4121-30 Khlebnikov A, Skaug T, Keasling JD, modulation of gene expression from the arabinose-inducible araBAD promoter,J Ind Microbiol Biotechnol. 2002 Jul;29(1):34-7.
This is suggested to be an adhesion responsive promoter. It contains the binding site for CpxR-P (phosphorylated CpxR). CpxR is one member of the two-component Cpx response to envelope stress. Briefly, envelope stress causes CpxA, an inner-membrane histidine Kinase to Phosphorylate CpxR, allowing it to bind to CpxR-P responsive regions and regulate downstream target genes. See 'Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway', Karen Otto and Thomas J. Silhavy, PNAS, Vol. 99., No, 4., 2287-2292. 2002.
This part comes from the genome of E.coli TR235, as used in the PNAS paper of Otto and Silhavy mentioned previously. TR235 is K-12 MC4100 containing a pCpxR-LacZ fusion inserted at the (Lambda)att site, plus some other modifications (see Otto and Silhavy). The part is a portion of the CpxR responsive region used in these LacZ fusions.
Hybrid promoter: HSL-LuxR activated, P22 C2 repressed
Hybrid promoter consisting of the Lux box which overlaps partly with -35 box. Binding sites for P22 C2 OR2 and OR1 are located between the -35 and -10 boxes and downstream of them respectively.
-35 box is partly composite. -10 is taken from the P22 phage PR regulatory region
OmpF promoter that is activated or repressesed by OmpR according to osmolarity.
Promoter OmpF is activated by phosphorylated OmpR at low osmolarity, and is repressesed by phosphorylated OmpR at high osmolarity.In Escherichia coli, osmoregulation is mediated in part by the actions of such a two-component system consisting of EnvZ and OmpR. These proteins act to control the relative levels of the outer membrane porin genes, ompF and ompC. At low osmo larity, OmpF predominates in the outer membrane, while at high osmolarity the OmpF porin is replaced by OmpC.OmpF has a larger pore and a faster ??ow rate than OmpC. Reference: K. Mattison, R. Oropeza, N. Byers, L. J. Kenney, J Mol Biol 315, 497 (Jan 25, 2002).
The osmolarity range of OmpF sensing still have to be confirmed
Promoter OmpF is activated by phosphorylated OmpR at low osmolarity, and is repressesed by phosphorylated OmpR at high osmolarity.In Escherichia coli, osmoregulation is mediated in part by the actions of such a two-component system consisting of EnvZ and OmpR. These proteins act to control the relative levels of the outer membrane porin genes, ompF and ompC. At low osmo larity, OmpF predominates in the outer membrane, while at high osmolarity the OmpF porin is replaced by OmpC.OmpF has a larger pore and a faster ??ow rate than OmpC.
PAI+LasR -> LasI+GFP & AI+LuxR --| LasI+GFP
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the policistronic expresion of LasI enzyme and GFP, double terminator
The biobricks BBa_K266005 and BBa_E0840 where used to build this biobrick.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Incompatible with host expressing cI repressor.
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
Positively regulated, OmpR-controlled promoter. This promoter is taken from the upstream region of ompF. Phosphorylated OmpR binds to the three operator sites and activates transcription.
The promoter includes three OmpR binding sites: F1, F2, and F3. The promoter starts at -107 and goes up to the transcriptional start site at +1. A distant fourth binding site, F4, has been deleted. It is involved in negative regulation of ompF (Head, Tardy and Kenney, 1998), so its deletion is intended to make the promoter solely activating. The efficacy of this promoter versus the ompC upstream region (BBa_R0082) or the truncated ompC upstream region (BBa_R0083) is currently unknown.
This is identical to BBa_R0051 exept that some base has been modifed to alow for additional ZFP repressors to bind
This part promotes the transcription of TyrRS(aminoacyl tRNA synthetase).
E.coli We synthetized it according to the GeneBank.
Promoter for transcribing keys to act on locks based on Isaacs, Collins, et. al: "Engineered riboregulators enable post-transcriptional control of gene expression" KP3 absorbs three nucleotides from mixed site ("TACTAGAG"), so that the key has a 5 nucleotide spacer region (i.e. "TAGAG") between the transcription start site and the first nucleotide of the key. We are still looking into what the optimal size for the spacer region is. See also KP2 (BBa_J01007), and Key1 (BBa_J01008), Key2 (BBa_J01009), Lock1 (BBa_J01010), and Lock2 (BBa_J01011).
Rock-paper-scissors - Paper secondary plasmid [lux pR, LacI generator]
iGEM2009 Team:Southampton rock-paper-scissors 'Rock' secondary plasmid.
Regulated expression of LacI (C0012) by Lux pR (R0062).
See relevant basic biobrick parts (R0062, B0034, C0012, B0015).
Promoter driving the lasI gene in Pseudomonas aeruginosa. Activated by LasR+3OC12HSL and not RhlR+C4HSL (Seed et al., 1995) ~4x stronger than the lasB promoter (BBa_R0079; Seed et al., 1995). Switch point ~0.1nM 3OC12HSL (Seed et al., 1995)
Promoter sequence cropped to 4nt upstream of Las box according to example of Whiteley and Greenberg, J. Bac, 2001
Genomic sequence of Pseudomonas aeruginosa
This part encodes E coli protein Ag43, with an additional 6xHis tag for easy purification and quantification. Ag43 expression is known to cause E coli cells to adhere together, and the 6xHis tag was added to allow for convenient study of variation of E coli aggregation with respect to Ag43 expression levels.
Ag43 is an outer membrane E coli protein, so it has a N-terminal 52 amino acid long signal peptide sequence, which is cleaved after Ag43 pro-protein is transported into the periplasmic space. Since strong dependence has been demonstrated on the signal peptide cleavage on the signal peptide-protein junction, the 6xHis tag should be added after Ala53, the residue immediately after the signal peptide sequence.
This comes from Gibson assembly of fragments from a previous biobrick, BBa_K1352000 by Team:Aberdeen_Scotland, 2014.
This device is regulated by UV irradiation
Overlap extension of synthetic oligonucleotides
dapAp is the promter of dapA gene in E.coli. DapA enzyme is part of diaminopimelate (DAP)biosynthesis pathway. DAP is necessary for peptidoglycan and lysine biosynthesis. dapAp prmoter has been previously studied: Acord04: Acord J, Masters M (2004). "Expression from the Escherichia coli dapA promoter is regulated by intracellular levels of diaminopimelic acid." FEMS Microbiol Lett 235(1);131-7. PMID: 15158272 It has been shown that dapAp driven expression is inhibited at hight DAP. We have tried to confirm this by
Ptet-rbs-rfp-Ter-Plac-rbs-tetR-Ter. It produces rfp when there is enough lacI or less lactose.
LacI Consensus Binding Site in sigma 70 binding region
Double Promoter (LuxR/HSL, positive / P22 cII, negative)
Promoter Family Member (U037O11+D014O22)
constitutive promoter with (TA)10 between -10 and -35 elements
With 10 TA repeats between the -10 and -35 elements, the distance between the two elements is optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'on' state.
Rock - Primary plasmid (part 1) [RhlR generator]
Constitutive promoter selected to allow for continuous expression of C0171.
See relevant basic biobrick parts (J23119, B0034, C0171, B0015)
this hybrid promoter is designed to be consecutively off.It combines with plasR(R0079) and lambda cI OR1(see C0051).Adding P-AI-1 sould activate the promoter, while repressed by lambda cI.
"CI binding of the OR region. It binds OR1 with highest affinity, OR2 with next highest, and OR3 with lowest"(http://www.nonanone.com/qw/files/Biology%204450/test2_lecture8,9.txt)
Ptet-rbs-rfp-Ter. It produces rfp when there is less tetR.
OmpR-P strong binding, regulatory region for Team Challenge03-2007
Contains the F1 consensus site from the OmpF regulator. Contains a LacI repressor consensus binding site between the -35 and -10 portions of a T7 core promoter region, and a TetR consensus binding site upstream of the -35 site.
The weaker binding repressor binds upstream of the core promoter (upstream of -35), while the stronger binds inbetween the -35 and -10.
Information for creation of this construct came from the following sorces: Yoshida et al. JBC 2006 Frank et al. JMB 1997 Rong et al. PNAS 1998 Merabet et al. Biochemistry 1995 Su et al. JBC 1998 Kedracka-Krok et al. JPC 1999 Kleinschmidt et al. Biochemistry 1988 Butler et al. JBC 1982 Jorgensen et al. JBC 1991 Hillen et al. JMB 1984 Head et al. JMB 1998
This E. coli promoter is positively regulated by arabinose. araC is the regulator protein, binding to arabinose prevents the repression of pBAD.
This part is similar to BBa_I0500, but it differs from that one in several point mutations in the sequence.
This promoter is repressed by lacI and luxR with HSL. It contains a lux box and a lacI binding site.
R0010 modified with the insertion of a lux box in between -35 and -10.
Dual-Repressed Promoter for LacI and LambdacI
This part is a new construct of a promoter that is dually repressed by either LacI or LambdacI proteins.
LambdacI usually binds to two operator sites, one located upstream from -35 and one located between -35 and -10. By itself, LacI binds downstream from -10. This made the part design very straightforward, since the locations of the operator sites do not overlap at all. In order to complete this part, we simply combined the proper sequences and we had a dually-repressed promoter.
This part was made by combining BioBrick parts R0010(LacI promoter) and R0051 (LambdacI promoter), along with intermediate sequence between these operator sites.
Promoter Family Member (U097NUL+D026O22)
A Double Reporter (LacZ-alpha and GFP) loaded on pSB3C5. For more information please refer to BBa_I732403.
The pR regulatory region is a 98 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two operator sites at which HK022 cI protein can bind cooperatively. The -35 and -10 regions from pR are included along with some flanking sequences. The cutoff for the 5' end of the regulatory region was placed just after the pM -10 region which promotes transcription of a gene upstream (thus the pM -10 region is not included). The cutoff for the 3' end of the regulatory region was placed immediately prior to the RBS.
Derived from STHK022N . Literature claims that there is no cross-talk between HK022 cI regulatory region, Lambda cI, 434 cI and P22 cI, but this has not been verified experimentally by the synthetic biology group at MIT.
Promoter driving the rhlI gene in Pseudomonas aeruginosa. Activated by the RhlR+C4HSL. Also likely activated by LasR+3OC12HSL
This part is from the bacteriophage M13 genome, bp 2716-2764 in M13K07. It directs transcription of M13 gene VI (BBa_M13506, BBa_M13006). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Promoter activated by RhlR in concert with C4-HSL. C4-HSL is produced by RhlI, and acts as a quorum-sensing autoinducer. This is the natural sequence taken from Pseudomonas aeruginosa. Crosstalk: this promoter is also activated at a low level by LasR with its associated HSL.
The -10 and -35 sites are labeled as identified in [Pearson97]. The part begins with the RhlR binding site, and (rather arbitrarily) ends with the first transcribed base (+1).
Pseudomonas aeruginosa rhlAB promoter
Promoter for cell division genes ftsI and ftsW
BBa_S03331is based on the reguatory region preceding cell division genes in the mra cluster and includes the biobrick head and tail restriction sites.
A promoter for the first nine genes of the Escherichia coli mra cluster of cell division and cell envelope biosynthesis genes, including ftsI and ftsW. Hara H, Yasuda S, Horiuchi K and Park JT. Journal of Bacteriology (1997) 179: 5802-5811
Lac system inverter controlling the expression of LasR
The biobricks BBa_Q04121, BBa_B0034 and BBa_K266002 where used to build this biobrick
Promoter Family Member (U037O11+D062O22)
Placement of the operator site was determined to optimize steric interference between C2003 and RNA polymerase.
This promoter was rationally designed and synthesized.
Promoter Family Member (U061O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732401.
Double Promoter (constitutive / TetR, negative)
Promoter Family Member (U073O11+D026O22)
Promoter Family Member (U097NUL+D050O22)
V.cholerae ctx gene promoter region. Transcription takes place In the presence of a ToxR dimer. We don't have the -10 and -35 sites annotated (need to do this).
GENBANK M21250
NOT Gate Promoter Family Member (D001O1wt1)
Promoter from the lack operon cloned in the reverse orientation.
Scissors - Primary plasmid (part 1) [LasR generator]
Part of the iGEM2009 Team:Southampton rock-paper-scissors 'scissors' primary plasmid.
Constitutive promoter selected to allow for continuous expression of C0179.
See relevant basic biobrick parts (J23119, B0034, C0179, B0015)
This is the PP promoter taken from the P2 phage genome. It is an inducible promoter that is activated by a class of activators, including P2 ogr (I746350), PSP3 pag (I746351) and phiR73 delta (I746352). These different activators should cause different levels of activity of the PP promoter.
Promoter, Standard (434 cI regulated)
[Note: This is the same part as R0052 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The 434 cI regulatory region sequence is a 89 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. 434 cI repressor protein, <bb_part>BBa_C0052<bb_part>, binds to it. This segment contains O-R1, O-R2, P-R, and P-RM -35.
Wild-type promoter modified to comply with BBa_S0001: TTGACA-17N-GATACT
Rock-paper-scissors - Scissors secondary plasmid [las pR, LacI generator]
iGEM2009 Team:Southampton rock-paper-scissors 'scissors' secondary plasmid.
Regulated expression of LacI (C0012) by Las pR (R0079).
See relevant basic biobrick parts (R0070, B0034, C0012, B0015).
PgolTS-golS-PgolB-rbs-tetR-Ter-PtetO-rbs-rfp-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter It is a test of our suicide circuit for gold ions.
PgolTS-golS-PgolB, tetR, PtetO, rfp, Plac, tetR, Pcons2, lacI, Ter
promoter to xylA without CRP and several binding sites for xylR
removed crp binding site removed rbs removed several xylR binding sites
Promoter Family Member (U073O11+D038O22)
The BBa_J23100 constitutive promoter with Lac systems inverter
The biobricks BBa_J23100 and BBa_Q04121 where used to build this biobrick
Double Promoter (lacI, negative / P22 cII, negative)
LasR/LasI Inducible & RHLR/RHLI repressible Promoter
The natural spacer between the -35 and -10 is 14bp and the natural O1 site for RHLR/RHLI is 20bp long. There was not much over lap of the -35 or the -10 with this sequence, so it was nessicary to chop off some of the binding site sequence in order to maintain the correct spacing and preserve the -35 and -10 sequence. The binding site sequence is missing a tcc on is 5' end and a t on its 3' end.
Assambly of [http://parts.igem.org/Part:BBa_J23104 J23104] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
PTAC promoter followed by an RBS, green fluorescent protein and stop codon, for testing the effectiveness of the PTAC hybrid promoter designed by iGEM2009 Team:Southampton.
Inclusion of the RBS and stop sequence to ensure correct functionality observed.
Assambly of [http://parts.igem.org/Part:BBa_J23119 J23119] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Equivalent part to r0040, but in BBb format. In house, part is referred to as pBca9145-Bca1091 {Ptet}. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
PCR ca1091F/R on pSB1A2-r0040 (~100 bp, EcoRI/BamHI) Sub into pBca9145-Bca1089 (EcoRI/BamHI) Product is pBca9145-Bca1091 ---- ca1091F Forward EcoRI/BglII for Ptet part ttctggaattcatgAGATCTtccctatcagtgatagag ca1091R Reverse BamHI for Ptet part GtttGGATCCgtgctcagtatctctatc
Promoter, Standard (p22 cII regulated)
[Note: This is the same part as R0053 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The p22 cII regulatory region sequence is a 97 base-pair sequence with the standard BioBrick prefix and suffix sections on its ends. p22 cII repressor protein, BBa_C0053, binds to it. This segment contains O-R1, O-R2, a fragment of O-R3, the -35 of P-RM, and P-R (-10 and -35 from Tom Knight)
Modified from wild-type to comply with BBa_S0001: TTGACA-17N-GATACT
This is an 'upside-down' version of I14032. It is a strong, constitual promoter. By adding this part, we wish to emphasise the possibility of engineering both DNA strands, and the additional reglatory possibility that this gives. The upstream sequence is non-functional and simply there to faciliate gel analysis or to physically enable the part to be recomineered (see the Edinburgh 2007 project where it is flanked by dif-sites).
When dealing with reverse complements of a locus, suffix and prefix need be fitted in opposite order.
This sequence is part of the E.coli K12 genome.
Promoter Family Member (U097NUL+D062NUL)
P_template1 serves as the truss for PCR cloning. By respectively applying 7 different primers upstream and downstream, that is, 49 pairs of primers in total, we have got 49 different promoters. Then we made them digsted and loaded into a vector that has already possessed a lacZ-alpha fragment. In this way, we can read from the color of the clones on the plate whether the inserted promoter works or not. By this means, We can try seven different positions for Operator A and seven for Operator B, thus constructing 7*7 kinds of promoters which have actually constituted a specific promoter family. After transformed into the four test plasmids (pCR00, pCR01, pCR10, pCR11) together with a downstream double-reporter system, we will get to know the solo-repression and co-repression effects of the two repressors on the specific promoter.
Promoter (luxR & HSL regulated -- lux pR)
Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file>Image1.gif</bb_file>" width="614" height="362">
Promoter Family Member (U085O11+D002O22)
NOT Gate Promoter Family Member (D001O11)
BBa_K091146 promoter (PAI+LasR inducible & AI+LuxR repressible) controls the expresion of LasI enzyme, no terminator
The biobricks BBa_K091146 and BBa_S03154 where used to build this biobrick
Promoter Family Member (U037O11+D062NUL)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3K5. For more information please refer to BBa_I732403.
Promoter Family Member with Hybrid Operator (D001O46)
The fhuA promoter. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of <PfhuA> basic part K112708 PCR of dv019/dv020 on MG1655 (225bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+225, L) Product is pBca1256-K112708 ----------------------------------------------- dv019 Forward biobricking of <PfhuA> cgataGAATTCatgAGATCTgctaagcgtgaaataccggatgg dv020 Reverse biobricking of <PfhuA> cgttaGGATCCactctgatgtaaagtgaatgataacg
P2 promoter in agr operon from S. aureus
The agr P2 operon is an autocatalytic sensory transduction system in Staphylococcus aureus. P2 promoter regulates the synthesis of AgrB (a transmembrane protein) and AgrD (precursor of AIP autoinducing peptide). AIP binds to AgrC, a membrane binding receptor and it phosphorylates AgrA. The phosphorylated AgrA increases the expression level from P2 promoter.
Codon usage has been optimised for E. coli (which also seems to give good results for B. subtilis
The section of the sequence constituting the P2 promoter is subject to rather more debate than the sequences of the coding regions. [http://jb.asm.org/cgi/content/full/186/22/7549 Koenig, Robbin L., Ray, Jessica L., Maleki, Soheila J., Smeltzer, Mark S., Hurlburt, Barry K. Staphylococcus aureus AgrA Binding to the RNAIII-agr Regulatory Region J. Bacteriol. 2004 186: 7549-7555] shows the areas of the sequence to which phosphorylated AgrA binds while L Rao, R K Karls, and M J Betley: In vitro transcription of pathogenesis-related genes by purified RNA polymerase from Staphylococcus aureus. Bacteriol. 1995 May; 177(10): 2609???2614. shows the transcription start sites; we took the overall extent of the promoter region from that labelled in Morfeldt, E., K. Tegmark, and S. Arvidson. 1996. Transcriptional control of the agr-dependent virulence gene regulator, RNAIII, in Staphylococcus aureus. Mol. Microbiol. 21:1227???1237. (not available online).
NOT Gate Promoter Family Member (D001O22)
Inverted tetR promoter with noncoding, spacer DNA downstream of it to make the total length 250 bp. The promoter faces the 5' direction.
This is a modified version of the LacIQ promoter tha should bind LacIQ tighter than the original.
Promoter Family Member with Hybrid Operator (D001O12)
Promoter Family Member (U073O26+D062O61)
Ptet-rbs-rfp-Ter-Plac-rbs-tetR-Ter-Pcons2-rbs-lacI-Ter. It produces rfp when there is less lactose.
Ptet, rfp, Plac, tetR, Pcons2, lacI, Ter
This is a adenylate cyclase promoter that has been reported to oscillate in cell cycle dependant manner.
Ref: J. Biol. Chem., Vol. 260, Issue 5, 3063-3070, 03, 1985 Transcription of the Escherichia coli adenylate cyclase gene is negatively regulated by cAMP-cAMP receptor protein H Aiba [bases -53 to 12]
PCR mv028/mv029 on rrnB native sequence (721bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+910, L) Product is pBca1256-K112118 ----------------------------------------------- mv028 Forward Biobricking of rrnB P1 promoter cgataGAATTCatgAGATCTatgcactaaaacgtatcctacgcccg mv029 Reverse Biobricking of rrnB P1 promoter cgttaGGATCCcgccttcccgctacagagtcaagc
LasR and 3OC12HSL regulated promoter with CI Operator 1 (binding site for lambda cI repressor)
Complex Quorum sensing circuit that receives the signal of PAI+LasR and AI+LuxR to control the production of LuxI and LasI enzymes.
The biobricks BBa_K266006 and BBa_K266000 where used to build this biobrick
Infrared signal reporter (J23119:IFP:J23119:HO1)
Gene sequence is obtained directly from Tsien's lab.
Promoter Family Member (U085O11+D062O22)
Rock-paper-scissors - Rock primary plasmid
Composite part, consisting of BBa_K180011 and BBa_K180012, but with BBa_K180011 reading along reverse strand, and BBa_K180012 reading along the forward strand. Forms the 'rock' primary plasmid in Team:Southampton's rock-paper-scissors project design.
This part has been specified as a basic BioBrick, due to some of the component parts reading in reverse (unable to specify using the component BioBrick format).
Double terminator + Constitutive promoter + Strong RBS
This part contains a double terminator and a constitutive prokaryotic promoter and a strong RBS (BBa_B0014 + BBa_J23100 + BBa_B0030). It is a simple promoter in the goals to begin gene transcription. For Sup???biotech-Paris (2009) team this part is used when adding to a recombinant lambda phage and to begin the transcription of synthetic genes inside the Lambda phage genome.
BBa_B0014 : Designed by Reshma Shetty Group: Registry (2003-07-16). BBa_J23100 : Designed by John Anderson Group: iGEM2006_Berkeley (2006-08-04.) BBa_B0030 : Designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. Group: NCBS, Prather Lab.
This promoter can be repressed by lacI protein and induced by either IPTG or arabinose.
IPTG has better effect on this promoter than arabinose
LsrA promoter (indirectly activated by AI-2)
The regulatory network for the uptake of Escherichia coli autoinducer 2 (AI-2) is comprised of a transporter complex, LsrABCD; its repressor, LsrR; and a cognate signal kinase, LsrK. This network is an integral part of the AI-2 quorum-sensing (QS) system. Once uptaken into the cell, AI-2 will be phosphorylated under the effect of LsrK. Without the presence of AI-2, LsrA promoter is inhibited by the repressor, LsrR. Phosphorylated AI-2 will prevent LsrR from inhibiting this promoter, hence activating it so that the downstream sequence can be expressed. To prevent the cell from self-inducing this promoter, we must suppress its AI-2 producing capability. It can be done by knocking out LuxS, one of the vital gene required for producing AI-2. By doing that, LsrA promoter can only be activated under the presence of AI-2 from other sources than the host cell which is LuxS mutant (say, by contact with normal cells). More information on AI-2 quorum-sensing (QS) system please refer to: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1952038
Constitutive Promoter, High Transcription
Promoter Family Member (U073O11+D050O22)
Promoter Family Member with Hybrid Operator (D001O47)
The placement of the operator was designed to be near optimal for steric interference between C2003 and RNA polymerase.
Anderson promoter J23102 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Inverted tetR promoter with noncoding, spacer DNA upstream of it to make the total length 450 bp.
Double Promoter (LuxR/HSL, positive / cI, negative)
Pars promoter from Escherichia coli chromosomal ars operon.
This is the promoter region of the Escherichia coli chromosomal ars promoter. It is repressed by ArsR in the absence of arsenate or arsenite (Cai, J., and DuBow, M.S. 1996. Expression of the Escherichia coli chromosomal ars operon. Canadian Journal of Microbiology 42, 662-671; Diorio, C., Cai, J., Marmor, J., Shinder, R., and DuBow, M.S. 1995. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in Gram negative bacteria. Journal of Bacteriology 177, 2050-2056).
No modifications were made to the sequence. There appears to be an unexpected repeat of ACT immediately before the SpeI site. This does not affect the biobrick suffix but means that the suffix is 5 bases downstream of the annotated transcriptional start site rather than 2 as recommended. The reason for this is unclear.
Escherichia coli K12. Sequence obtained from X80057 (gi:510824). Regulatory sequence annotation from Cai, J., and DuBow, M.S. 1996. Expression of the Escherichia coli chromosomal ars operon. Canadian Journal of Microbiology 42, 662-671; Diorio, C., Cai, J., Marmor, J., Shinder, R., and DuBow, M.S. 1995. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in Gram negative bacteria. Journal of Bacteriology 177, 2050-2056.
Promoter Family Member with Hybrid Operator (D001O16)
Assambly of [http://parts.igem.org/Part:BBa_J23118 J23118] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
Allows for regulation of following gene with laqIq and IPTG...laqIq in same plasmid represses the promoter and IPTG proportionally depresses the promoter (Boer, et al., 1983).
Derived from trp and lac promoter sequences.
A inducible promoter plus a strong RBS B0034, repressed by TetR, induced by tetracycline and aTc.
This part should be used along with TetR.
B0034 is based on Elowitz repressilator.
Weaker version of wild type pBAD ( I13453 ).
Site-directed mutagenesis on I13453 with the following primers: Forward: TAATCTTATGGATAAAAAAGCTATGGCATAGC Reverse: GCGGATCCTACCTGACGCTTTTTATC
E. Coli CreABCD phosphate sensing operon promoter
CreABCD phosphate sensing operon promoter taken from RegulonDB.
Promoter Family Member (U037O11+D002O22)
Promoter Family Member (U037O11+D026O22)
This is the pBAD promoter (BBa_I13453) in the opposite orientation. It can be used to drive transcription in the direction of suffix to prefix.
Rock - Primary plasmid (part 2) [tac pR, mCherry and LasI generator]
Regulated expression of LasI (C0178) and mCherry (K180008) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180008, B0015)
constitutive promoter which outputs .4 TIPs point mutants of wild type rrnB promoter P1
NOT Gate Promoter Family Member (D001O44)
This is identical to BBa_I12007 exept that some base has been modifed to allow for additional ZFP repressors to bind.
Promoter Family Member with Hybrid Operator (D001O26)
Double terminator + constitutive promoter
This part contains a double terminator and a constitutive prokaryotic promoter. It is a simple promoter to begin gene transcription. For Sup???biotech-Paris (2009) team this part is used after to be added to a recombinant lambda phage to begin the transcription of synthetic genes inside the Lambda phage genome.
BBa_B0014 : Designed by Reshma Shetty Group: Registry (2003-07-16) BBa_J23100 : Designed by John Anderson Group: iGEM2006_Berkeley (2006-08-04)
The recA(SOS)promoter gene from the chromosomal DNA isolated from MG1655 has been PCR amplified and inserted into plasmid pSB1A2(part number BBa_B0015).
Promoter, Standard (lambda cI regulated)
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross Drew Endy
[Note: This is the same part as R0051 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. The cI regulated promoter is based on the pR promtoer from bacteriohage lambda. The promoter has two two DNA binding sites for lambda cI repressor <bb_part>BBa_C0051</bb_part>. cI binding results in repression of transcription. The specific sequence used here is based on the cI repressible promoter used in the Elowitz repressilator (and references therein).
In order to address concerns about the promoter transcribing in the reverse direction, we have removed the -35 and -10 signals responsible for the promoter activity in the reverse direction. (More details needed here! DE, 2/24/03) Modified to comply with BBa_S0001: TTGACA-17N-GATACT Incompatible with host expressing cI repressor.
CinR, CinL and glucose controlled promotor
This promotor is controlled by cinr, cinl and extrcellular glucose concentration. High extracellular glucose concentration will inhibit this promotor through the action of phosphorylated OmpR. Phosphorylated OmpR will bind the OmpR binding site of the promotor and block the movement of RNA polymerase. In contrast, the combined action of CinR and CinL will turn on the promotor. But only when the extracellular glucose level is low so OmpR dephosphorylate and dissociate from its binding site will this promotor become active. In essence, this promotor function as an AND gate, it will turn on if and only if the extracellular glucose level is low and there are some functional CinR and CinL protein inside the cell.
The key to the function of the promotor is the inhibitory ability of OmpR binding site. To ensure maximum affinity with phosphorylated OmpR, we use all three OmpR binding site.s.
Double Promoter (cI, negative / TetR, negative)
Promoter Family Member (U061O11+D038O22)
Promoter Family Member (U049O11+D062O22)
Promoter Family Member with Hybrid Operator (D001O24)
Constitutive promoter J23100 with Lac system inverter controlling the expression of LasR
The biobricks BBa_J23100 and BBa_K266003 where used for build this biobrick
This pLas' promoter contains the regulatory region upstream of the LasI gene in Pseudomonas aeruginosa. K091117 includes a LasR binding site as well as a -10 region modified from CATAAA to TATAAA to reflect our pLasXOR promoter design. Activation is expected to occur in the presence of the autoinducer PAI-1 and the LasR protein. The pLas' promoter serves as a control for the evaluation of the pLasXOR promoter, part BBa_K091118.
To minimize leaky transcription, a secondary transcriptional start site was excluded from the design.
The sequence of the LasI upstream regulatory region is available at http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=176640
K091157 is a modified version of the pLux' promoter (BBa_K091156) containing the addition of a LasR binding site between the -35 and -10 regions. This promoter is expected to be activated in the presence of AI-1 and LuxR and repressed in the presence of PAI-1 and LasR.
In order to fit the LasR binding site between the -35 and -10 regions, the last base of the Las Box was cropped and the -35 region was modified to overlap with the first two bases of the Las Box.
The LasR binding site is from the region upstream of the LasI gene in Pseudomonas aeruginosa. The rest of the promoter is the same as BBa_K091156.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
position 3 mutated promoter lux pR-3 (luxR & HSL regulated)
The position 3 mutated promoter lux pR-3 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 3 mutated promoter lux pR-3 gives a higher expression of downstream genes in an autoinduction system. In another word, the lux pR-3 has a higher efficiency than the lux pR. Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
In the promoter lux pR's sequence, the C residue of position 3 was changed to a T using PCR method.
The position 3 mutated promoter lux pR-3 is based on promoter lux pR (BBa_R0062).
Promoter Family Member (U097O11+D002O22)
Promoter Family Member (U097O11+D062O22)
mutant promoter of lacI gene, which enables high level of lacI protein expression
point mutation made from E. Coli genomic lacI gene promoter
pcI (for positive control of pcI-lux hybrid promoter)
constitutive promoter with (C)12 between -10 and -35 elements
With 12 C repeats between the -10 and -35 elements, the distance between the two elements is optimal for the sigma factor to bind to the promoter. Thus, the promoter should be in the 'on' state.
This promoter is the right promoter in the lux cassette of Vibrio Fischeri. There is weak constitutive expression of downstream genes. Expression of downstream genes can be up-regulated by the action of the LuxR activator protein in complex with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein can form a complex with two molecules of the AHL. This causes a conformational change in the protein, allowing it to bind to a site on the lux promoter and induce transcription.
This part was obtained by taking the reverse complement of R0062, ordering appropriate oligos, then annealing and extending them.
This hybrid promoter uses the LsrA promoter that is capable of being repressed by LsrR - induction can occur with phospho-AI-2. Binding sites for cI repressor also occur. As a result the promoter is off in the absence of AI-2 and on in the presence of AI-2, but always off in the presence of cI.
The promoter region for LsrA should remain unaltered while cI binding sites (OR1 and OR2) are introduced downstream. The OR2 sites with cI bound should not allow transcription. The cI binding sites have a natural 6 bp spacer between them. It would be desirable to position the cI binding sites immediately downstream of the -10 region of the LsrA promoter. However, experimental evidence for the transciption start (+1) appears to be lacking.
The Missouri Western/Davidson team designed primers for LsrA and extracted the DNA from E. coli MG1655. The CI portion was pulled from the registry as part number R0051.
Assambly of [http://parts.igem.org/Part:BBa_J23102 J23102] and lacI-binding-site of [http://parts.igem.org/Part:BBa_R0010 R_0010] The "lacI binding site" of Part [http://parts.igem.org/Part:BBa_R0010 R_0010] differs from lacO from Part [http://parts.igem.org/Part:BBa_J33207 BBa_J33207].
optimized (A) repeat constitutive promoter with 17 bp between -10 and -35 elements
NOT Gate Promoter Family Member (D001O66)
This part has the arabinose regulated promoter (AraC/pBAD) upstream of a ribosome binding site. This is ideal for inserting a protein to be expressed downstream of this part as it's expression can be regulated by the presence of the concentration of arabinose in the growing environment/media of the cells. ==What this part actually does== This composite part includes the AraC/pBAD promoter and a strong ribosome binding site designed from the Anderson family of RBS. The AraC/pBAD promoter can be used with prokaryotic chassis to regulate expresion of proteins. When the AraC/pBAD promoter is placed upstream of your canditate translational unit (protein to be expressed) it allows control of the trancription of the said protein. This is done via controlling the concentration of arabinose sugar in the environment/solution of bacterial growth. Controlling the transcription will not ensure translation. The ribosome binding site is ideally placed a small distance away from the promoter site to ensure that the ribosome will bind and start translating the protein coding sequence found downstream.
The part was designed with RFC10 assembly from supplied biobricks of the iGEM2009 distribution.
The AraC promoter can be found naturally at 1.52min of the E.coli genome ([http://ecogene.org/geneinfo.php?eg_id=EG10054 EcoGene number EG10054]. The RBS was taken from the designed sequences of RBS by the Anderson family. For more information visit the original part page [http://partsregistry.org/Part:BBa_J61100 here].
An intermediate part designed to be used with the AND gate designed by Caltech iGEM '10: [http://2010.igem.org/Team:Caltech/Project Project Details].
Designed to be placed upstream of positive feedback mechanisms and desired output genes.
The Registry of Standard Biological Parts
Promoter Family Member (U097NUL+D002O22)
A Double Reporter (LacZ-alpha and GFP-AAV) loaded on pSB3C5. For more information please refer to BBa_I732401.
Positively regulated, OmpR-controlled promoter. This promoter is derived from the upstream region of ompC. Phosphorylated OmpR binds to the operator site and activates transcription. This is a truncated version of BBa_R0082.
The promoter deletes the C2 and C3 regions of BBa_R0082 (bases 37-74 in BBa_R0082) and inserts an 8-base linker before the -35 and -10 signal region. The efficacy of this promoter versus the full ompC upstream region (BBa_R0082) or the ompF upstream region (BBa_R0084) is currently unknown. The a at base 41 is mutated to t to avoid XbaI site. Speculation that this was a previous assembly site.
Promoter Family Member (U061O11+D014O22)
Promoter Family Member (U061O11+D050O22)
This part contains the pLac promoter and a ribosome binding site (BBa_R0010 + BBa_B0030). The Lac promoter is constitutively activated, but it can be repressed by the Lac I repressor from E.coli. This promoter is really useful to control a gene expression by inactivating it with LacI repressor. When LacI is absent the gene should be expressed, and in the presence of LacI, pLac is inhibited. So this part is really suitable for gene expression control by inactivation of pLac with LacI. We can use this part to control a failed feedback mechanism which induces the Lambda phage lytic cycle. In E.Coli, pLac is a part of the lactose operon. LacI is constantly expressed and represses the pLac. In the presence of Lactose, LacI is inhibited and pLac is active and allows the transcription of beta-galactosidase. Lactose can be substituted by IPTG (Isopropyl β-D-1-thiogalactopyranoside). This part already contains a strong RBS to begin directly a gene transcription.
Part:BBa_R0010 Part:BBa_B0030, Designed by Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003. Group: NCBS, Prather Lab.
Scissors - Primary plasmid (part 2) [tac pR, mBanana and LuxI generator]
Regulated expression of LasI (C0178) and mBanana (K180009) by tac pR (K180000), controlled by presence of lac/IPTG or trp in the cell.
See relevant basic biobrick parts (K180000, B0034, K180009, B0015).
Promoter Family Member (U097O11+D062NUL)
promoter to xylF without CRP and several binding sites for xylR
removed RBS crp binding and xylR binding sites
This promoter is a modified version of the Mnt promoter that is also responsive to LacI. The promoter should be repressed by Mnt repressor. It should also be repressed by LacI, and in the absence of Mnt repressor, should be induced by IPTG.
Mnt repressor binds as a tetramer to two half-operator sites. Introduction of a LacI binding site between the Mnt promoter -10 sequence and the start site for transcription should allow for repression by LacI. Accordingly, the hybrid promoter was designed by 1) Remove all bases of mnt promoter 3??? to -10: gagtcgtattaattt will be replaced by tgtgtggaattgtga, and 2) Position lacI binding site (composed of an inverted repeat) from the lacI regulated promoter (R0010) immediately 3??? to truncated mnt promoter.
This part comes from the Mnt promoter and the LacI promoter
Promoter of flhDC gene from E. coli flagella
''flhDC'' is the only class 1 gene that is part of the construction of the flagella. It is the master regulator of the biosynthesis of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). The promoter of ''flhDC'' is regulated by many transcription factors, for instance, OmpR* and EnvZ* are two repressors of this gene. '''Work in progress :''' Characterization of the repression by OmpR* and EnvZ*
The PCR on colony to amplify the promoter of ''flhDC'' did not work. We amplified both the promoter and the gene and then we did a specific amplification of the promoter from the full operon. We had a PCR amplicon of the right length this time. The primers used to amplify the promoter and the gene are : * Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTGTATGTGCGTGTAGTGACGAGTACAG * Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTATTATTAAACAGCCTGTACTCTCTGTTCATCC Then to amplify only the promoter we used : * Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGTTGTATGTGCGTGTAGTGACGAGTACAG * Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTATCCCACCCAGAATAACCAACTTTAT
Promoter Family Member (U049O11+D026O22)
Promoter of flgB gene from E. coli flagella
''flgB'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGACAGTATCGCGATGATCGCCACGCTACG *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAAGCATATCTCCTCCGCAGGTATCAAAATT
Regulated by the presence of Iron and UV light irradiation
This part is from the bacteriophage M13 genome, bp 4055-4103 in M13K07. It directs transcription of M13 gene IV (BBa_M13504, BBa_M13004). Its identity as a promoter was described in Gene (1980) 11:129-148 based on -10,-35 homology and fragment of genome able to bind RNAP.
Anderson promoter J23101 without RFP in backbone vector pSB1C3 to easily fuse the promoter with other reporters e.g. the lux operon.
Promoter of flhB gene from E. coli flagella
''flhB'' is a class 2 gene that is part of the construction of the flagella ( ''Ordering Genes in a Flagella Pathway by Analysis of Expression Kinetics from Living Bacteria'' - S. Kalir, J. McClure, K. Pabbaraju, C. Southward, M. Ronen, S. Leibler, M. G. Surette, U. Alon ). Its promoter is consitutively repressed. It is activated by FlhD4C2 and maybe by FliA with different strength ( ''Using a Quantitative Blueprint to Reprogram the Dynamics of the Flagella Gene Network'' - Shiraz Kalir and Uri Alon ). '''Work in progress :''' Deeper quantitative characterization
The primers used to amplify this sequence are : *Forward : GTTTCTTCGAATTCGCGGCCGCTTCTAGAGCCACGTCATATCAGGCGGTCTGATAAGG *Reverse : GTTTCTTCCTGCAGCGGCCGCTACTAGTAGTTTTGTCGTCGCTCTCGTCAGACACGTC
Promoter Family Member (U097O11+D026O22)
Phns in BBb1 format. ---- This part is in BBb Format. It is flanked by BamHI and BglII sites instead of XbaI and SpeI. More information about the BBb Format is available at: [http://openwetware.org/wiki/Template:AndersonLab:BBb_Standard BBb Standard Description Page]
Construction of [<Phns>] Basic Part K112701 PCR dv001/dv006 on Phns from E. coli K12 MG1655 (~342 bp, gp = A) PCR dv005/dv003 on Phns from E. coli K12 MG1655 (225 bp, gp = B) PCR dv004/dv002 on Phns from E. coli K12 MG1655 (~149 bp, gp = C) --------------------------------------------------- PCR dv001/dv003 on A+B (355 bp, gp = D) --------------------------------------------------- PCR dv001/dv002 on C+D (697bp, EcoRI/BamHI/DpnI) Sub into pBca1256 (EcoRI/BamHI, 2472+697) Product is pBca1256-K112701 ----------------------------------------------- dv001 Forward Biobricking of [<Phns>] CgATAgaattcATGagatctGAAATATAGCTGTGCCATCAGCC dv002 Reverse Biobricking of [<Phns>] cagtcggatccGCACGAAGAGTACGGATG dv003 Removing the 2nd EcoRI site in [<Phns>] (R) GCCAGGAATGTAAGGgATTCAAAATTGTTC dv004 Removing the 2ND EcoRI site in [<Phns>] (F) GAACAATTTTGAATcCCTTACATTCCTGGC dv005 Removing the FIRST EcoRI site in [<Phns>] (F)CGCTTAATAGGGgATTCTCGTAAACAC dv006 Removing the FIRST EcoRI site in [<Phns>] (R)GTGTTTACGAGAATcCCCTATTAAGCG
promoter of phoB in E.coli, an external phosphate regulated promoter which can be activated when encountering phosphate limitation.
The promoter region should contain PhoB binding site
This is the promoter activated by the transcriptional regulator XylR (BBa_I723136) in response to detection of environmental xylene.
Transciptional regulator reliant on screams of someone, somewhere in trouble.
DNA sample - Taken from Clark Kent's Coffee Mug at the Daily Planet
Promoter, Standard (luxR and HSL regulated -- lux pR)
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr Drew Endy
[Note: This is the same part as R0062 except that the -10 and -35 sites and spacing have been changed to comply with BBa_S0001]. Promoter activated by LuxR in concert with HSL The lux cassette of V. fischeri contains a left and a right promoter. The right promoter gives weak constitutive expression of downstream genes.This expression is up-regulated by the action of the LuxR activator protein complexed with the autoinducer, 3-oxo-hexanoyl-HSL. Two molecules of LuxR protein form a complex with two molecules of the signalling compound homoserine lactone (HSL). This complex binds to a palindromic site on the promoter, increasing the rate of transcription.
This promoter is based on the Vibrio fischeri quorum sensing gene promoters. Two genes LuxI and LuxR and transcribed in opposite directions as shown below. The original sequence from which the parts <bb_part>BBa_R0062</bb_part> and <bb_part>BBa_R0063</bb_part> were derived is shown in the picture below. <img src="<bb_file part=BBa_R0062>Image1.gif</bb_file>" width="614" height="362"> Modified to comply with BBa_S0001: TTGACA-17N-GATACT
we synthesize the OR promoter by replacing OR3 binding site of lambda CI with OR1 binding site of CI434.
making sure CI434 protein can repress CI's transcription.
similar to lambda lysis/lysogenic regulatory region.
Right facing promoter (for xylF) controlled by xylR and CRP-cAMP
potential looping feature possibly removed
removed RBS, left promoter, and several xylR binding sites
position 3&5 mutated promoter lux pR-3/5 (luxR & HSL regulated)
The position 3&5 mutated promoter lux pR-3/5 is a product of site-directed mutagenesis of promoter lux pR (BBa_R0062). The promoter lux pR is based on the Vibrio fischeri quorum sensing gene promoters. The position 5 mutated promoter lux pR-3/5 gives a lower expression of downstream genes in an autoinduction system. In another word, the lux pR-3/5 has a lower efficiency than the lux pR(BBa_R0062). Similar to promoter lux pR, the expression is up-regulated by the action of LuxR protein combined with the signal molecule HSL.
We have generated two point mutants by site-directed mutagenesis from the sequence of lux pR(BBa_R0062). In these mutants, the G residue of position 5 was changed to a C and the C residue of position 3 was changed to a T respectively.