gfasPurple, purple chromoprotein reporter system (Strong Promoter, Strong RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943002 with chromoprotein gfasPurple(BBa_K1033919) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene. For detailed characterization data, see the experience page.
spisPink, Pink chromoprotein reporter system (medium Promoter , weak RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943007 with chromoprotein spisPink(BBa_K1033932) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene. For detailed characterization data, see the experience page.
This part encodes a blue chromoprotein named aeBlue. This part is useful for reporter.
The part has been codon-optimised for expression in E. coli using http://sg.idtdna.com/CodonOpt
The aeBlue sequence from Uppsala team 2012 (BBa_K864401) was chemically synthesised.
This chromoprotein, cjBlue, naturally exhibits strong green/blue color when expressed. Compared to amilCP(BBa_K592009) and amilGFP(BBa_K592010), the color development is slower. On agar plates and in liquid culture, the color is readily visible to naked eye after 24-48 hours of incubation. The DNA was codon-optimized for expression in E.coli and synthesized by the Korean company Bioneer Corporation.
Codon-optimization for expression in E.coli necessary.
This chromoprotein from the coral Stylophora pistillata, spisPink, naturally exhibits color when expressed.
Synthesized and codon optimized for E coli by GenScript.
gfasPurple, purple chromoprotein reporter system (medium Promoter , weak RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943003 with chromoprotein gfasPurple(BBa_K1033919) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene. For detailed characterization data, see the experience page.
This chromoprotein from the coral Galaxea fascicularis, gfasPurple, naturally exhibits strong color when expressed.
GoldenGate compatible AmilCP, Blue chromoprotein
A variation of the part BBa_K592009 that has been made compatible with the GoldenGate MoClo Assembly Standard as it is free from BsaI and BpiI recognition sequences. The chromoprotein naturally exhibits strong blue color when expressed. Codon-optimised for expression in dicotyledenous plants.
Domesticated for GoldenGate by removal of internal BsaI and BpiI recognition sequences. Codon-optimised for expression in dicotyledenous plants.
fwYellow, yellow chromoprotein reporter system (Strong Promoter, Strong RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943000 with chromoprotein fwYellow(BBa_K1033910) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene.
spisPink, Pink chromoprotein reporter system (Strong Promoter, Strong RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943006 with chromoprotein spisPink(BBa_K1033932) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene. For detailed characterization data, see the experience page.
This part encodes a chromoprotein, aeBlue, with LAA degradation tag
The aeBlue sequence was codon-optimised using http://sg.idtdna.com/CodonOpt. The degradation tag is based on Edinburgh 2014.
The BBa_K1685005 part is given LAA-degradation tag designed by Edinburgh 2014 team.
This chromoprotein naturally exhibits strong color when expressed.
This construct is synthetic. It was synthesized from a template where the consensus sequence for the "color dependancy" in order to obtain a chromoprotein with a unique yellow color.
tsPurple, purple chromoprotein (incl RBS)
Cloned from TinselPurple vector, DNA 2.0.
MLC Glucose Sensitive Yellow Chromoprotein Construct
This composite part features our MLC2 construct combined via 3A assembly with a yellow chromoprotein+RBS part (BBa_K1033931). During transcription, the MLC2 binding site is bound by the protein MLC, inhibiting production of yellow chromoprotein. As glucose concentration increases in the cell, more MLC is uptaken and bound to the membrane by a mechanism involving the transmembrane glucose permease called IICBGlu. This means that higher glucose concentrations subsequently result in higher expression levels of the yellow chromoprotein and thus makes it a glucose-inducible chromoprotein producing part. Specifically, this part tests the hypothesis for how MLC affects the inducibility of a strong promoter when placed downstream of the base promoter sequence.
One issue to consider was where to place the MLC binding site and what strength promoter to use as a base. This was solved by creating multiple variants of the promorer (MLC1-4). This part may be improved by considering how the RBS is affected by the position of the MLC binding site.
1. Plumbridge, Jacqueline. ???DNA Binding Sites for the MLC and NagC Proteins: Regulation of nagE, Encoding the N-Acetylglucosamine-Specific Transporter inEscherichia Coli.??? Nucleic Acids Research 29.2 (2001): 506???514. Print. 2. http://parts.igem.org/Part:BBa_J23110 3. http://parts.igem.org/Part:BBa_K1033931
This chromoprotein from the coral Montipora efflorescens, meffBlue, naturally exhibits strong color when expressed.
This chromoprotein, amilGFP, naturally exhibits very strong yellow color when expressed. The color is strong and readily visible to naked eye both in LB-culture and on agar plates. The DNA was provided by Jeffrey Miller at UCLA. It was made BioBrick-compatible after removal of two illegal internal restriction sites (EcoRI and PstI).
Two illegal internal restriction sites (EcoRI and PstI) was removed.
eforRed, Red chromoprotein reporter system (medium Promoter , weak RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943005 with chromoprotein eforRed(BBa_K592012) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene. For detailed characterization data, see the experience page.
AmilCP (BBa_K592009) compatible with Genabler assembly
AmilCP (BBa_K592009) part with inward facing EarI recognition sites at the ends. It is compatible with Genabler assembly.
To be compatible with Genabler assembly, the part has inward-facing EarI sites at the ends. When assembled by BioBrick assembly, five N-terminal residues MetAlaSerSerSer- and one C-terminal Ala residue will be added to the original sequence of BBa_K592009.
AmilCP is based on the BioBrick BBa_K592009.
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
Synthesized and codon optimized for E. coli by Bioneer Corp.
J23110-B0034-aeBlue (chromoprotein) (E.coli optimized)
aeBlue is a blue chromoprotein extracted from the basal disk of a beadlet anemone Actinia equina. The protein has an absorption maximum at 597nm and a deep blue colour. The protein aeBlue is of the GFP family with significant sequence similarities. This part is a functionally active variant of BBa_K864401.
The promoter J23110 is sufficiently strong to yield expression levels making the blue protein clearly visible and eventually opaque.
The protein was discovered, extracted and analyzed in (Shkrob, 2005). It was synthesized by Bioneer Corporation, codon optimized for E.coli.
This Biobrick is created by putting enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 under constitutive promoter R0011. Enzyme cassette CrtEBIY (with individual rbs) of Part BBa_K274200 converts colourless farnesyl pyrophosphate to orange beta-carotene (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). Sequence and Features
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that this Biobrick works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (lycopene) production than strain TOP10.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. Please refer to Part BBa_K274200 for more information on individual components.
This composite part features our MLC2 construct combined via 3A assembly with a yellow chromoprotein+RBS part (BBa_K1033931). During transcription, the MLC2 binding site is bound by the protein MLC, inhibiting production of yellow chromoprotein. As glucose concentration increases in the cell, more MLC is uptaken and bound to the membrane by a mechanism involving the transmembrane glucose permease called IICBGlu. This means that higher glucose concentrations subsequently result in higher expression levels of the yellow chromoprotein and thus makes it a glucose-inducible chromoprotein producing part. Specifically, this part tests the hypothesis for how MLC affects the inducibility of an average strength promoter when placed downstream of the base promoter sequence.
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs), Part BBa_K118005 (CrtI with rbs) and Part BBa_K118013 (CrtY with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB, CrtI and CrtY convert colourless farnesyl pyrophosphate to orange beta-carotene (via intermediates geranylgeranyl pyroiphosphate, phytoene and lycopene). The orange beta-carotene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006, BBa_K118005 and BBa_K118013 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274210) and arabinose-inducible promoter I0500 (see Part BBa_274220), transformed and tested in E.coli strain MG1655. Amount of beta-carotene produced can be measured by photospectrometer with absorbance at 455nm (beta-carotene extraction using acetone). A related Composite Biobrick is Part BBa_K274100 (CrtEBI with rbs), which "stops" at one step before beta-carotene, producing (and retaining) red lycopene pigment.
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that this Biobrick works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (beta-carotene) production than strain TOP10.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006, Part BBa_K118005 and Part BBa_K118013.
This Composite Biobrick is created by standard assembly of Part BBa_K118014 (CrtE with rbs), Part BBa_K118006 (CrtB with rbs) and Part BBa_K118005 (CrtI with rbs), which are submitted by previous iGEM teams. The whole cassette is on plasmid pSB1A2 (high copy, Ampicillin resistance). Together, enzymes CrtE, CrtB and CrtI convert colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). The red lycopene pigment can then be used as a coloured signal output, e.g. for biosensors. There is already individual ribosome binding site before each enzyme gene sequence. Internal restriction sites have been removed by previous iGEM teams. Please refer to Parts BBa_K118014, BBa_K118006 and BBa_K118005 for more information on individual Biobricks components. This Composite Biobrick has been put under constitutive promoter R0011 (see Part BBa_K274110) and arabinose-inducible promoter I0500 (see Part BBa_274120), transformed and tested in E.coli strain MG1655. Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).
The colourless precursor, farnesyl pyrophosphate, is naturally produced in E.coli using Non-mevalonate Pathway (from pyruvate and glyceraldehyde-3-phosphate). Our experiences showed that the Composite Biobrick, when put under a promoter, works better in E.coli strains with up-regulated level of precursors. E.coli strain MG1655 demonostrates higher level of pigment (lycopene) production than strain TOP10.
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. This Composite Biobrick is created by standard assembly of Part BBa_K118014, Part BBa_K118006 and Part BBa_K118005.
fwYellow, yellow chromoprotein reporter system (medium Promoter , weak RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943001 with chromoprotein fwYellow(BBa_K1033910) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene.
This chromoprotein eforRed naturally exhibits red color when expressed. The color is weaker than RFP, however. On agar plates and in liquid culture, the color is readily visible to naked eye in less than 24 hours of incubation. The DNA was codon-optimized for expression in E.coli and synthesized by the Korean company Bioneer Corporation.
eforRed, Red chromoprotein reporter system (Strong Promoter, Strong RBS)
Team Uppsala 2012 chromoprotein attracts many interests because it's more convenient than fluorescent protein for it can be observed with naked eyes. However, characterized data of these proteins are few. Because a single coding region is on the plasmid, we constructed BBa_K1943004 with chromoprotein eforRed(BBa_K592012) this year and did characterization. We've constructed a series of plasmids which are all in the same pattern: a strong/weak promoter, a strong/weak RBS and a chromoprotein. We want to monitor the speed of expression of these plasmids in normal incubation conditions (like 37℃ overnight for LB agar plate and 37℃ 180rpm for LB broth). The expression speed and strength of the constructed biobricks will be carefully monitored and gave others a relative scale for using chromoprotein as a reporter gene. For detailed characterization data, see the experience page.
This part encodes a chromoprotein, aeBlue, with AAV degradation tag
AeBlue chromoprotein could be expressed as a deep blue color in E.coli, which was added by Uppsala University in 2012. We add lva tag to before stop codon to increase the degradation of the Aeblue chromoprotein, which is necessary in our earth map for color changing.
Chromoprotein is extracted from Uppsala University in 2012.
This chromoprotein, amilCP, naturally exhibits very strong color when expressed. The color is blue/purple and is visible to naked eye, thereby requiring no instruments to observe. This DNA was provided by Jeffrey Miller at UCLA. It was made BioBrick-compatible after removal of one illegal internal restriction site (EcoRI).
Illegal internal restriction site had to be removed (EcoRI).
yA (green pigment) from Aspergillus nidulans
The gene yA from Aspergillus nidulans encodes a p-diphenol oxidase (laccase) that converts a yellow pigment precursor to the mature green form. The green pigment is located in the walls of the conidia and protects the spores from damage by ultraviolet light. A yA mutant produces yellow spores.
Compatible with the Plug'n'Play assembly standard
This composite part features our MLC2 construct combined via 3A assembly with a yellow chromoprotein+RBS part (BBa_K1033931). During transcription, the MLC2 binding site is bound by the protein MLC, inhibiting production of yellow chromoprotein. As glucose concentration increases in the cell, more MLC is uptaken and bound to the membrane by a mechanism involving the transmembrane glucose permease called IICBGlu. This means that higher glucose concentrations subsequently result in higher expression levels of the yellow chromoprotein and thus makes it a glucose-inducible chromoprotein producing part. Specifically, this part tests the hypothesis for how MLC affects the inducibility of an average strength promoter when placed upstream of the base promoter sequence.
This Biobrick is created by putting enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274100 under constitutive promoter R0011. Enzyme cassette CrtEBI (with individual rbs) of Part BBa_K274110 converts colourless farnesyl pyrophosphate to red lycopene (via intermediates geranylgeranyl pyroiphosphate and phytoene). Amount of lycopene produced can be measured by photospectrometer with absorbance at 475nm (lycopene extraction using acetone).
Gene coding sequences come from Pantoea ananatis (formerly Erwinia uredovora) (Accession number D90087), a type of Gram negative Enterobacteriaceae. Please refer to Part BBa_K274100 for more information on individual components.
AeBlue chromoprotein could be expressed as a deep blue color in E.coli, which was added by Uppsala University in 2012.
This chromoprotein from the coral Stylophora pistillata, spisPink (also known as spisCP), naturally exhibits strong color when expressed. The protein has an absorption maximum at 560 nm giving it a pink color visible to the naked eye. The strong color is readily observed in both LB or on agar plates after less than 24 hours of incubation. The protein spisPink has significant sequence homologies with proteins in the GFP family.
Stylophora pistillata. The protein was first extracted and characterized by Alieva et. al. under the name spisCP (GenBank: ABB17971.1). This version is codon optimized for E coli by Genscript.
This chromoprotein from the coral Anemonia majano, amajLime, naturally exhibits strong color when expressed.