Vikram Vijayan, Allen Hsu, Lawrence Fomundam
I13307.E0430 Lac-Regulated Cin Amplifier with YFP Reporter
Aditi Shrivastava, Jiwon Lee, Sarah Welch
Strong RBS (B30), EYFP + LVA (E32) LINKS OK
Victoria Chou, Kenneth Nesmith, Madeleine Sheldon-Dante
Olmecs: Oscillator 2 of Synchro-Dualator
This half bit contains the integrase/excisionase for HK022 and attP/attB rev comp for P21. The attachment sites flank a unidirectional terminator. This half bit is designed to work with the P21 half bit (BBa_I11063)
Built by Josh as an intermediate in screening plasmid construction.
I13306.E0430 Tet-Regulated Las Amplifier with YFP Reporter
LacI QPI regulated by lambda cI with EYFP
YFP output used to calibrate pBAD promoter (titrate with arabinose)
Trans-splicing Tetrahymena ribozyme to remove LVA tag
Designed ribozyme to remove LVA tag from constructs built by Reshma containing a LVA tag on the YFP gene. Replace the LVA tag with a stop codon to allow for higher expression.
The ribozyme was designed for trans-splicing based on the design principles in Kohler et al 1999. The target sequence is a EYFP gene with a LVA degradation tag. The target splice site is the U closest to the end of the EYFP. The IGS (P1 helix) of the ribozyme was redesigned to bind to this region and an extended antisense region to the LVA tag of around 50 bp was added. The spliced on 3' exon is simply a stop codon. The expected behavior is for the LVA tag to be replaced by a stop codon, allowing for a return to the normal degradation rate for the protein (i.e. long life) and thus higher expression. Contains the same lac controlled promoter R0011 and double terminator used in the target construct.
Intermediate in two promoter screening plasmid
Barkai-Leibler design experiment part B (434cI)
This forms the repressor of a Barkai-Leibler oscillator. The 434 cI has no LVA tag (BBa_I12014 is the same as BBa_C0052 with no LVA tag).
This part to work in conjunction with BBa_I12000. This part creates one of the proteins involved in the Modified Barkai-Leibler relaxation oscillator design (repressor protein: 434 cI). In the presence of Lambda cI, this part will create 434 cI, which will in turn repress the production of Lambda cI in BBa_12000. The LVA tag was removed from the 434 cI coding region due to the fact that we want the repressor to be as potent as possible, thus, if it doesn't degrade as quickly, the relative strength should be increased.
Alvin Carter Powers, Sara Neves (Fighting Darwins)
RBS, araC coding region, terminator, araC binding region araC positively regulates output of TIPs
p(Ltet0-1), Medium RBS, LacI + LVA, Weak RBS, Lambda CI + LVA, Strong RBS, EYFP + LVA
Olmecs: Repressilator version 2 (Lambda cI)
The P21 half bit uses integrase/excisionase from P21 and attP/attB (rev comp) from HK022. The attachment sites are flanking a unidirectional terminator. This half bit is designed to be used with the HK022 half bit (BBa_I11062)
Deletion of the C at position 1194. This is right at the end of I0500, after the transcriptional start of the downstream gene but before the RBS. Shouldn't make a difference, but needs to be checked out.
LacI ts (mut 265) QPI regulated by lambda cI with mCherry
CheR behind a tet promoter and Elowitz RBS
PoPS->PoPS amplifier (LasR-based, HSL driven)
LasR protein generator upstream of pLas. Useful device for cell-cell signaling. Expected to require HSL for maximum PoPS out.
Test of BBa_R0011 (LacI regulated) using YFP
LuxR receiver strain w/ LuxR regulation (Pbad)
Lux R receiver with the YFP reporter. Same as I13034, but with TetR regulator under Pbad promoter.
pLac controlled receiver/reporter device (LuxR-based, HSL driven)
Composite part based on the I13262 protein generator. LacI regulated promoter activated luxR-based HSL receiver. pLux-R is driving a YFP output device.
FecA'-GFP under inducible promoter and rbs
A protein (lambda cI) subassembly for modified BL oscillator based on I12036
This part produces POPS output to connect with a reporter.
Intermediate in screening plasmid construction
EYFP coding device with promoter, RBS, and terminator. No degradation tag.
EYFP with promoter, RBS, and terminator. No degradation tag.
R0011.I13277 Lac-Regulated Cin Amplifier
aiiA (LVA-) protein generator driven by pBad
Useful for measuring the ability of aiiA to degrade HSL in a cell expressing LuxI
Constitutive aiiA & dsRed using P(Lac) IQ
p(Lac) IQ, Medium RBS, aiiA + LVA, Strong RBS, dsRed - LVA
Lambda cI857 (Lambda II) QPI regulated by 434 cI with EYFP
Identical to BBa_I5100 except the LVA tag has been removed.
Lambda cI QPI regulated by 434 cI with mCherry
σx Generator + PcomFA-E0240
Ng Siu Wang Edward, Jason Jang, Joo Min Jung
SigmaX generator, SigmaX-inducible Phelicase encoding GFP
Both comX gene and Phelicase are found in genomic sequence of Streptococcus pneumoniae NCTC7465 strain, while the RBS+GFP+DTT, downstream of PCelA, is BBa_E0240. The promoter encoding comX is BBa_K880005 and the double terminator for it is BBa_B0015
David Pohlman, Blythe Ferriere, Aile Able, Erica McFarland
Our part consists of an OMP-R binding region that regulates our reporter gene, eYFP. The OMP-R binding region is an omp-c operator, which has three OMP-R binding sites. Our part is regulated by the ENVZ-OMPR two-component regulatory system. ENVZ is a osmolarity sensor on the inner membrane and phosphorylates OMP-R in the presence of high osmolarity. When phosphorylated, OMP-R binds to the omp-c operator and recruits RNAP to transcribe the downstream reporter gene. Fluorescence intensity due to the production of eYFP is a measure of the system's activity. When all three binding sites are occupied by phosphorylated OMP-R proteins, RNAP cannot bind to the DNA and transcribe downstream. This part can be used as an osmolarity indicator. Our team used this part as a glucose sensor because the presence of glucose changes osmolarity. Different concentrations of glucose produce different, quantifiable fluorescence intensities.
Our team had to combine the genes in a specific order. First our team cut out and purified the eYPF gene. We added this gene to a plasmid containing RBS. Next we cut out the RBS+eYFP intermediate and ligated them into the plasmid containing OMP-R. The RBS and OMP-R parts were too small to run out and extract on a gel because they would run off of the end of the gel. Therefore, we had to start the process with eYPF.
This BioBrick was created from parts found in the Registry. These parts were BBa_R0082, BBa_B0032, and BBa_E0030.
The Elowitz Repressilator design, optimized for construction with quadpart inverters. YFP output device on same plasmid as repressilator.
completed 2 color temperature sensor, tetracycline tuneable
2 color temperature sensor, with regulatory system decoupled from color production and under the control of a tetracycline repressor for tuning
Olmecs: Repressilator version 4 (P22 c2)
p(Lac) (R10), Strong RBS (B30), LasR+ LVA (C79)
434 cI QPI regulated by Lambda cI with EYFP
Olmecs: Repressilator version 3 (434 cI)
I13304.E0430 Tet-Regulated Rhl Amplifier with YFP Reporter
R0011.I13279 Lac-Regulated Las Amplifier
Same as J06006, but also expresses LVA+ variant of LuxI.
p(Bla), Strong RBS, aiiA + LVA, Strong RBS, dsRed - LVA
p(Ltet0-1), Strong RBS, LacI + LVA, Weak RBS, Lambda CI + LVA, Strong RBS, EYFP + LVA
σx Generator + PcelA-E0240
Jordy Evan, Nadia Benedicta, Angeline Widjaja
SigmaX generator, SigmaX inducible PCelA encoding GFP
Both comX gene and PCelA are found in genomic sequence of Streptococcus pneumoniae NCTC7465 strain, while the RBS+GFP+DTT which is downstream of PCelA is BBa_E0240. The promoter encoding comX is BBa_K880005 and the double terminator for it is BBa_B0015
Reporter containing LacZ alpha fragment. Requires complementation with omega fragment. (See notes on E0033)
R0040.I13277 Tet-Regulated Cin Amplifier
Same as the other TetR heterodimer test construct (I12232), but with different promoters in front of the two components of the heterodimer.
GFP inserted in front of Pbad CFP. Intermediate in screening plasmid construction.
This is meant to serve as a reporter device for the synchronized oscillator.
This part generates the chez protein which is responsible for the dephosphorylation of cheY protein in bacteria flagella movement. A 6-histidine tail is added to the chez coding region. The pTet promoter can be repressed by the TetR repressor protein(C0040). It is constitutively on without the presence of TetR repressor.
pLac controlled receiver (LuxR-based, HSL driven)
Composite based on the I13262 PoPS amplifier. Lac regulated, no downstream output device
A protein (lambda cI) subassembly for modified BL oscillator based on I12006
This part has POPS output to connect with reporter. It is based on I12006.
R0040.I13271 Tet-Regulated Rhl Amplifier
434 cI QPI regulated by Lambda cI with mCherry
This is a test construct for the two-hybrid system.
QPI-optimized Elowitz Repressilator. YFP output device w/o ssra tail. (R0040.Q04510.Q04121.Q04400.E0430)
Strong RBS (B30), LasR + LVA (C79) LINKS OK
R3 protein + GFP reporter to indicate expression of protein R3
A protein (lambda cI) subassembly for modified BL oscillator based on I12040
This is a composite part of a T7 promoter and a dedicated RBS. It will be used to construct a reporter device for VM1.0
p(Ltet0-1), Strong RBS, LacI + LVA, Weaker RBS, Lambda CI + LVA, Strong RBS, EYFP + LVA
LacI ts (mut 241) QPI regulated by lambda cI with mCherry
I13305.E0430 Lac-Regulated Las Amplifier with YFP Reporter
Olmecs: Reporter for Repressilator versions
Wildtype lac promoter regulating expression of ECFP and EYFP (both +Clontech RBS, -LVA. Polycistronic message.)
LacI QPI regulated by lambda cI with mCherry
Lux I/R Sender and Receiver w/ TetR regulation
Ring Oscillator Synchronization Device (I13260.Q04121.I0461)
This is a device for synchronizing ring oscillators, based on research by Elowitz.
Lambda cI857 (Lambda II) QPI regulated by 434 cI with mCherry
LacI ts (mut 241) QPI regulated by lambda cI with EYFP
IPTG and arabinose are used to control expression of lambda cI/LVA and 434 cI, respectively. These two repressors bind into I12036 and modulate expression of YFP/AAV. Must be used with LacIq strain.
This part links together generators for lambda cI and 434 cI with I12036 and YFP reporter.
Aditi Shrivastava, Sarah Welch, Jiwon Lee
Barkai-Leibler design experiment part B (p22cII)
This forms the repressor of a Barkai-Leibler oscillator.
half bit contains integrase and excisionase coding regions for lambda phage and attachment sites for p22 int/xis proteins that flip a unidirecitonal terminator
p(Las) (R79), Strong RBS (B30), EYFP + LVA (E32)
Blue Chromoprotein + 2a + HBsAg + Yeast Secretion Tag + ADH1 Terminator
Brian Yang, Jonathan Krog, Sunil Deochand, Jonathan Kelly
Should make YFP when given AHL. (Used to be a twin of I13272; revised 6/27 to use E0432 instead of E0434.)
p(Lac), Medium RBS, TetR + LVA, Strong RBS, ECFP + LVA
Tet-repressible polycistronic CFP/YFP under B0034
LacI ts (mut 265) QPI regulated by lambda cI with EYFP
Same as J06007, but with LVA- version of LuxI.
434 cI regulatable expression of YFP (LVA-)
Identical to BBa_I5120 except the LVA tag has been removed.
Identical to BBa_I5120 except the LVA tag has been removed. It was removed using a hack in which I simply amplified the everything in the plasmid except the tag and did a blunt end ligation.
Ring Oscillator Synchronization Device (I13260.Q04510.I0461)
Lambda cI QPI regulated by 434 cI with EYFP
Wildtype lac promoter regulating expression of CFP (Clontech RBS, no ssra tail.)
A screening plasmid using YFP and CFP. The initial Pbad promoter has two extra restriction sites, HindIII and NcoI.
Lambda cI QPI regulated by LacI with EYFP
Alvin Carter Powers, Yong-Su Jin (Fighting Darwins)
RBS, lacI w/o degradation tag coding region, terminator, lacI binding region used in switch
input is luxI direct negative feedback by CI, integral feedback through p22 CII (in concert with part BBa_J1103). Generates bursts of gfp activity in response to step increases in luxI concentration. Should exhibit perfect(?!) adaptation.
Intermediate in building second version of screening plasmid. Pbad-RBS-CFP-HindIII-NcoI-RBS-YFP
Use this part to insert any 5?????TR of your gene of interest to screen for downregulation of your gene with small RNA. PCR amplify your 5???UTR with EcoRI and BamHI and clone into any backbone carrying this part. Digest whis part with EcoRI and BamHI and as RFP is replaced, this part functions as a red/white screening system. This allows you to create a reporter system with a fluorescent marker for screening for functional small RNAs. SYFP2 BBa_K864100 is situated downstrean of the RFP BBa_J04450 and is connected in frame with a 12 aa glycine-serine repeated linker BBa_J18922 , to prevent folding distruptions in the YFP or the 5???UTR. The first 6 nt of the glycine-serine linker is a BamHI restriction site (GGATCC), which translates into glycine-serine in E.Coli. When screening for working small RNAs, it's recommended to include the first 15 codons of the gene of interest.
Constructed from SYFP2 BBa_K864100 , linker BBa_J18922 and RFP BBa_J04450
p(Ltet0-1), Medium RBS, LacI + LVA, Weaker RBS, Lambda CI + LVA, Strong RBS, EYFP + LVA
Ring Oscillator Synchronization Device (I13260.Q04400.I0461)
Tet repressible promoter and dedicated RBS
This composite part is composed of a tet repressible promoter and a dedicated RBS. It will be used to construct a reporter system for VM1.0. GFP production from the reporter system can be compared to the GFP production of a similar system that uses a T7 promoter instead of an E. coli promoter and to I7102 which uses a slightly different dedicated RBS.
R3 protein -Terminator (to be fused to e.g. reporter)
B protein (434 cI) subassembly for modified BL oscillator
This part produces 434 cI (B) in presence of lambda cI (A).
p(Lac), Strong RBS, TetR + LVA, Strong RBS, ECFP + LVA
Light responsive system, dual regulation
Pbad regulated phycobilins (ho1 and pcyA) followed by Ptet regulated light sensor (Cph8). This should allow repressed Pbad to give low levels of accessory proteins and derepressed Ptet to crank out lots of light sensor protein
Tet promoter, Elowitz RBS, CheY coding. CheY is used in chemotaxis.
PoPS->PoPS amplifier (RhlR-based, HSL driven)
RhIR protein generator upstream of pRHL. Useful device for cell-cell signaling. Expected to require HSL for maximum PoPS out.
Lambda cI857 (E07700) QPI regulated by 434 cI with EYFP
IPTG and arabinose are used to control expression of lambda cI/LVA and 434 cI, respectively. These two repressors bind into I12040 and modulate expression of YFP/AAV. Must be used with LacIq strain.
This part links together generators for lambda cI and 434 cI with I12040 and YFP reporter.
(New and improved) Test circuit for I12006
IPTG and arabinose are used to control expression of lambda cI/LVA and 434 cI, respectively. These two repressors bind into I12006 and modulate expression of YFP/AAV. Must be used with LacIq strain.
This part links together generators for lambda cI and 434 cI with I12006 and YFP reporter.
This is an example of the 2nd version of the screening plasmid test. In this version there is only one copy of pBAD and the POPSin reads-through a CFP coding region before reaching the device.
Has a deletion of the C at base 1194 (at the end of I0500). This is after the transcription start of the Pbad promoter and before the RBS of the transcript, so it should not be significant.
R0040.I13279 Tet-Regulated Las Amplifier
RBS, cI-857 coding region, terminator, cI-857 binding region repressor inverter used in heat trigger
completed 2 color temperature sensor with rfp (low) bias
2 color temperature display system, with stronger low temperature color signal coupled with a less strong high temperature signal (rfp bias)
polycistronic CFP/YFP expression cassette
A single mRNA containing CFP and YFP under the strong RBS B0034 is driven by the tet repressible promoter, R0040.
R0011.I13271 Lac-Regulated Rhl Amplifier
LuxR generator + LacI regulated lambda cI (LVA)+LuxI (LVA)
Polycistronic lambda cI and LuxI proteins regulated by Lac Repressor (LacI). Downstream is consitutive LuxR expression cassette. System flanked by dual transcription terminators. System incompatible with TetR.
This is the wild-type cassette for kanamycin resistance.
Lambda cI857 (E07700) QPI regulated by 434 cI with mCherry
cI coding protein on plasmid backbone J61002
This is the wild-type cassette for ampicillin resistance.
I13303.E0430 Lac-Regulated Rhl Amplifier with YFP Reporter
input is luxI output is gfp negative feedback through cI Increases in level of luxI will cause transient pulses in cI, with levels returning to near zero asymptotically
I13308.E0430 Tet-Regulated Cin Amplifier with YFP Reporter
Lambda cI QPI regulated by LacI with mCherry
This is a testing construct for the TetR - TetR-4C heterodimerization system.
half bit contains integrase and excisionase coding regions for p22 phage and attachment sites for lambda int/xis proteins that flip a unidirecitonal terminator