This chromoprotein from the coral Anemonia majano, amajLime, naturally exhibits strong color when expressed.
Synthesized and codon optimized for E coli by GenScript.
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.
RFP from Discosoma striata (coral) with LAA-ssrA degradation tag (wt)
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LAA-ssrA degradation tag (part BBa_M0050). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYALAA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LAA tag is E. coli wild type ssrA tag and is reported to lead to very fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; Protein stability; Km of binding to the protease; Temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012)
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction site. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (AAV in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0050, but same tag was also used in paper by Andersen et al., (1998).[2]
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.
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.
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.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 15 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
AsPink chromoprotein in GoldenGate standard
AsPink chromoprotein in BBa and MoClo standard.
The AsPink is now compatible with both standards and can be used more effectively.
The chromoprotein was taken from the iGEM registry and altered to conform to the MoClo standard.
This chromoprotein from the coral Stylophora pistillata, spisPink, naturally exhibits color when expressed.
Contains an IPTG inducible promoter an RBS, RFP, and terminator.
cjBlue chromoprotein in BBa and MoClo standard
The cjBlue chromoprotein is now compatible with both standards and can be used more effectively.
AmajLime chromoprotein in BBa and MoClo standard
The AmajLime chromoprotein is now compatible with both standards and can be used more effectively.
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.
RFP from Discosoma striata (coral) with LVA-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added LVA-ssrA degradation tag. The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. This tag is commonly attached to repressor proteins for use in various gene networks (e.g., oscillators). The tag encodes peptide sequence AANDENYALVA and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The LVA tag is reported to lead to fast protein degradation, degrading GFP with rate -0.018 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: ClpXP and ClpAP protease and SspB mediator concentrations; protein stability; Km of binding to the protease; temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
The tag was attached to RFP using PCR and MABEL (mutagenesis with blunt-end ligation), thus avoiding introduction of additonal residues and restriction sites. Different parts of the tag are recognized by different proteins, for example, the final 3 residues (LVA in this case) are recognised by ClpX, whereas first 4 residues of the tag are required for efficient SspB binding.[1] Thus modifications of these critical residues alter the efficacy with what different proteases bind to it.
RFP comes from part BBa_E1010, tag sequence was obtained from paper by Andersen et al., (1998).[2]
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 6 AA linker as spacer (BBa_K1680000). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
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.
tsPurple chromoprotein in GoldenGate standard
tsPurple chromoprotein in BBa and MoClo standard
The tsPurple chromoprotein is now compatible with both standards and can be used more effectively.
EforRed chromoprotein in BBa and MoClo standard
The EforRed chromoprotein is now compatible with both standards and can be used more effectively.
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.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 12 AA linker as spacer (BBa_K1680002). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 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).
RFP from Discosoma striata (coral) with DAS-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part BBa_E1010) with added DAS-ssrA degradation tag (part BBa_M0052). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYADAS and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The DAS tag is reported to have low affinity to ClpX thus its mediated degradation very much depends on the concentration of SspB (ClpX mediator).[1] However, be aware that exact protein degradation rate is influenced by multiple other factors: ClpXP and ClpAP protease concentrations, protein stability, Km of binding to the protease, temperature [3]. References: [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3] Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part BBa_E1010, tag sequence was obtained from part BBa_M0052, but same tag was also used in paper by Andersen et al., (1998).[2]
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.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 9 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
This chromoprotein from the coral Galaxea fascicularis, gfasPurple, naturally exhibits strong color when expressed.
This circuit is composed of the constitutive promoter and photosensitive protein gene ccaS . ccaS protein is membrane protein. After being activated by 520-nm light, it could transfer its phosphoryl group to the intracellular protein ccaR,and this process could be inhibited by 650-nm light
It's not difficult to construct this part,but the strength of the promoter and the RBS will influence the abundance of protein ccaS,and finally influence the whole TCS.And there was a SpeI site and a EcoRI site,so we designed two silent mutations.The upstream of gene ccaS called sll1472 is of unknown function.
Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems
AeBlue chromoprotein in BBa and MoClo standard
The AeBlue chromoprotein is now compatible with both standards and can be used more effectively.
AeBlue0034 chromoprotein in BBa and MoClo standard
tsPurple, purple chromoprotein (incl RBS)
This chromoprotein naturally exhibits strong color when expressed.
Cloned from TinselPurple vector, DNA 2.0.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with intermediate 6 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
Synthesized and codon optimized for E. coli by Bioneer Corp.
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.
AeBlue chromoprotein could be expressed as a deep blue color in E.coli, which was added by Uppsala University in 2012.
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.
Protein coding sequence for a mutant (K145N) of the fluorescent protein Dronpa, codon optimized for Yeast. The mutation leads to photoswitchable monomerisation/multimerisation behaviour (as described by Zhou et al. 2012).
RFP from Discosoma striata (coral) with AAV-ssrA degradation tag
Mutant RFP from Discosoma striata (coral) (see part E1010) with added AAV-ssrA degradation tag (part I11012). The tag increases RFP turn-over rate, thus providing better temporal resolution of red fluorescence. In the same time, maximal fluorescence amplitudes will be lower as newly formed protein is degraded as soon as it is formed. The tag encodes peptide sequence AANDENYAAAV and is recognized by ClpA and ClpX unfoldases and ClpX mediator SspB.[1] ClpA and ClpX then form a proteosome-like complex with ClpP protease and the protein is degraded.[1] The final three residues of the tag determines the strength of interaction with ClpX and thus the final protein degradation rate.[2] The AAV tag is reported to lead to reasonably fast protein degradation, degrading GFP with rate -0.012 per minute.[2] However, be aware that exact protein degradation rate depends on multiple factors: 1) ClpXP and ClpAP protease and SspB mediator concentrations; 2) Protein stability; 3) Km of binding to the protease; 4) Temperature [3]. [1] Flynn, J. M. et al. Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584???9 (2001). [2] Andersen, J. B. et al. New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64, 2240???6 (1998). [3]Purcell, O., Grierson, C. S., Bernardo, M. Di & Savery, N. J. Temperature dependence of ssrA-tag mediated protein degradation. J. Biol. Eng. 6, 10 (2012).
RFP comes from part E1010, tag sequence was obtained from part I11012, but same tag was also usead in paper by Andersen et al., (1998).[2]
This circuit is composed of the constitutive promoter, ho1 chromoprotein and pcya chormoprotein. They facilitate Membrane protein ccaS and cph8 to be activated by light of certain wavelength.
Protein ho1 and protein pcyA have to combine with each other to function, so we put them behind the same promoter.
This part is from paper "Refactoring and Optimization of Light-Switchable Escherichia coli Two-Component Systems", and obtained from iGEM team HZAU's lab
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 15 AA linker as spacer (BBa_K1680003). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
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.
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.
AmilCP chromoprotein in BBa and MoClo standard
The AmilCP chromoprotein is now compatible with both standards and can be used more effectively.
Protein coding region for a fusion construct of Cre Recombinase and 145N Dronpa with 12 amino acid linker. Oligomerisation of Dronpa should stop Cre from accessing DNA in the nucleus thereby inhibiting it until light deactivation of dronpa.
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.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral) with barcode
Constantin Ahlmann-Eltze, Charlotte Bunne, Magdalena Büscher, Jan Gleixner, Max Horn, Anna Huhn, Nils Klughammer, Jakob Kreft, Elisabeth Schäfer, Carolin Schmelas, Silvan Schmitz, Max Waldha
This part is essentially identical to <partinfo>BBa_E1010</partinfo>, but has a point mutation in the barcode. Barcodes are discontinued, but composite parts using this part will have the mutated barcode.
Protein coding region for a fusion protein of Cre recombinase (BBa_K1680007) that is C- & N-terminal flanked with 145N Dronpa (BBa_K1680006) using a 9 AA linker as spacer (BBa_K1680001). The construct has a N-terminal NLS sequence (BBa_K1680004). In the expressed construct the two dronpa domains should form multimerize leading to an inactive Cre recombinase that is reversibly activatable through dronpa illumination (see Zhou et al 2012).
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 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 Montipora efflorescens, meffBlue, naturally exhibits strong color when expressed.
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.
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.
AmilGFP chromoprotein in BBa and MoClo standard
The AmilGFP chromoprotein is now compatible with both standards and can be used more effectively.
This part is a positive selection system for recombineering. It contains a kanamycin resistance gene Aph(3???)-I (aminoglycoside 3???-phosphotransferase, truncated version) with its native promoter as well as amilCP, a deep blue chromoprotein. amilCP gene is under the control of a strong constitutive promoter with a strong RBS to make it more intense than previous versions. The cassette is designed to be a double positive selection marker for recombineering. Once amplified with primer bearing homology to the targeted region, the cassette can be integrated in the genome through lambda red recombination. Once integrated, the colonies should become resistant to kanamycin and become pale blue overtime. However, the blue color is actually faint and really slow to become apparent.
The amilCP chromoprotein synthesis could be improved for the blue color to be visible in single copy faster. It has been of great concern through-out all the project and that is why we have put the strongest combination of promoter and RBS we have found to be well characterised. Still, expression in single copy is not so intense. In our case, amilCP can still be useful as a marker for plasmid background detection during recombineering experiment. Using only the kanamycin marker for recombineering, the blue colonies would represent plasmid transformants and not good recombinants. This use is good for us though, since background is a great concern in recombineering techniques, where screening can be long.
The part was constructed using a kanamycin resistance gene from plasmid pOK12 (available commercially) and amilCP from BBa_K592009. The promoter and RBS P1U8 were synthesised and come from Mutalik et al. (Nature 2013).