For the characterisation of the red light responsive system, under tetR regulation
Chimeric Cph1 light receptor/EnvZ (Cph8) protein requires phycocyanobilin (PCB) biosynthetic genes for PCB formation (ho1 and PcyA). The exposure to red light (660nm) inhibits the activity of the EnvZ histidine kinase domain. In the dark, the EnvZ histidine kinase phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. This part must be used in E.coli deficient in wild-type EnvZ. The heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) are under a constitutive promoter. Ho1 and PcyA are genes required for phycocyanobilin (PCB) biosynthesis and TetR binds to the p(TetR) (Part:BBa_R0040) that regulated light sensor (Cph8). aTc (anhydrotetracycline) binds to TetR and allows Cph8 production. The RFP is positively regulated by OmpR-controlled promoter. Phosphorylated OmpR binds to the operator sites and activates transcription of RFP. This is a test Biobrick for the red light responsive system with the RFP protein as output.
heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) under const
Two genes required for phycocyanobilin (PCB) biosynthesis and the coding region for the TetR protein with Ribosome Binding Site (RBS) under a strong promoter. The Part:BBa_K1132012 was inserted behind BBa_K608003, a strong promoter with a medium RBS. ho1 produces biliverdin IXalpha, it represents the first of two steps in PCB biosynthesis. PcyA converts biliverdin IXalpha to PCB. PCB associates with EnvZ fusion protein, Cph8 (Part:BBa_I15010), to transduce a light signal into a genetic response. TetR binds to the p(TetR) (Part:BBa_R0040) and inhibits its operation. It regulates the light sensor Cph8 (Part:BBa_K1132015). aTc (anhydrotetracycline) binds to TetR and allows Cph8 production. Both of these two parts construct the red light responsive system, under tetR regulation.
Promoter (OmpR, positive) followed by RFP protein generator and Cph8 (Cph1/EnvZ fusion) under TetR r
Chimeric Cph1 light receptor/EnvZ protein requires phycocyanobilin (PCB) biosynthetic genes for PCB formation (ho1 and PcyA). The exposure to red light (660nm) inhibits the activity of the EnvZ histidine kinase domain. In the dark, the EnvZ histidine kinase phosphorylates endogenous OmpR, a transcription factor which activates transcription from the OmpC promoter. This part must be used in E.coli deficient in wild-type EnvZ. The promoter p(TetR) is constitutively on and repressed by TetR (Part:BBa_P0440). TetR binds to the p(TetR) and inhibits its operation. It regulates Cph8 (Part:BBa_K1132015) production. Tetracycline or aTc (anhydrotetracycline) binds to TetR and allows Cph8 production. This part is an intermediate Biobrick for the characterisation of the red light responsive system (Part:BBa_K1132017) with the RFP protein as output. The monomeric RFP is a Red Fluorescent Protein with an excitation peak at 584 nm and an emission peak at 607 nm. The RFP is positively regulated by OmpR-controlled promoter. Phosphorylated OmpR binds to the operator sites and activates transcription of RFP. This must be used with Part:BBa_K1132013 composed of the heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR) under constitutive promoter.
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.
Stephen Lee, Roshan Kumar, Joe Levine, Ziyan Chu (Polkadorks, IAP 2004)
Lutz, R., Bujard, H., Nucleic Acids Research (1997) 25, 1203-1210.
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.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman
PoPS in -> RFP out Strong RBS (efficiency=0.6). Artificial 39 bp terminator (efficiency=0.99).
heme oxygenase (ho1), ferredoxin oxidoreductase (PcyA) and tetracycline repressor (TetR)
Two genes required for phycocyanobilin (PCB) biosynthesis and the coding region for the TetR protein. ho1 produces biliverdin IXalpha, it represents the first of two steps in PCB biosynthesis. PcyA converts biliverdin IXalpha to PCB. PCB associates with EnvZ fusion protein, Cph8 (Part:BBa_I15010), to transduce a light signal into a genetic response. TetR binds to the p(TetR) (Part:BBa_R0040) and inhibits its operation. It regulates the light sensor Cph8 (Part:BBa_K1132015). aTc (anhydrotetracycline) binds to TetR and allows Cph8 production.
The cph8 was taken from Tabor's pJT122 plasmid.
This is the red light sensor Cph8 with RBS.
Since Cph8 was considerably longer than the RBS added, sequencing was necessary to confirm the construct.
RBS.3 (medium) -- derivative of BBa_0030
Weak1 RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0030</bb_part>, <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("RBS-2" in figure 4-14 of thesis). Contact info for this part: Brian Chow
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
**highly** engineered mutant of red fluorescent protein from Discosoma striata (coral)
Campbell et al., PNAS v99 p7877 URL
monomeric RFP: Red Fluorescent Protein. Excitation peak: 584 nm Emission peak: 607 nm
TAATAA double stop codon added (DE). Four silent mutations made to remove three EcoRI sites and one PstI site: A28G, A76G, A349G, G337A.
RBS.1 (strong) -- modified from R. Weiss
Strong RBS based on Ron Weiss thesis. Strength is considered relative to <bb_part>BBa_B0031</bb_part>, <bb_part>BBa_B0032</bb_part>, <bb_part>BBa_B0033</bb_part>.
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix ("orig" in figure 4-14 of Ron Weiss thesis). No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info Brian Chow
modified E. coli thr terminator, replaced all A-T pairs in stem with C-G pairs
Artificial terminator, estimated %T~>90% *8bp stem, 6nt loop *Bidirectional, estimated reverse %T~>90%
Bidirectional, with the reverse estimated to be less effective than the forward. Has a polyA tail of 9 residues.
heme oxygenase (ho1) and ferredoxin oxidoreductase (PcyA)
Two required genes for phycocyanobilin (PCB) biosynthesis. ho1 produces biliverdin IXalpha, it represents the first of two steps in PCB biosynthesis. PcyA converts biliverdin IXalpha to PCB. PCB associates with EnvZ fusion protein, Cph8 (Part:BBa_I15010), to transduce a light signal into a genetic response.
RBS (Elowitz 1999) -- defines RBS efficiency
Varies from -6 to +1 region from original sequence to accomodate BioBricks suffix. No secondary structures are formed in the given RBS region. Users should check for secondary structures induced in the RBS by upstream and downstream elements in the +50 to -50 region, as such structures will greatly affect the strength of the RBS. Contact info for this part: Brian Chow
This part is identical to BBa_I15010, the cph8 red light sensor. We were unable to transform BBa_I15010 from the the 2011 iGEM distribution kit. PCR attempts using VF2 and VR failed to amplify any DNA from the well sample. In the end, we chose to amplify cph8 from Jeffrey Tabor's pJT122 plasmid provided by Chris Voigt. The cph8 in pJT122 contained an internal PstI site, which was removed by point-mutagenesis. The resultant DNA sequence is identical to that of BBa_I15010.
The source DNA (pJT122) contained illegal restriction site (PstI) that had to be removed.
Derived from the TE terminator of T7 bacteriophage between Genes 1.3 and 1.4 <genbank>V01146</genbank>.
Transcription terminator for the E.coli RNA polymerase.
Suggested by Sri Kosuri and Drew Endy as a high efficiency terminator. The 5' end cutoff was placed immediately after the TAA stop codon and the 3' end cutoff was placed just prior to the RBS of Gene 1.4 (before AAGGAG). Use anywhere transcription should be stopped when the gene of interest is upstream of this terminator.
Transcriptional terminator consisting of a 64 bp stem-loop.
tetracycline repressor from transposon Tn10 (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999)
Coding region for the TetR protein without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. TetR binds to the pTet regulator (BBa_R0040). aTc (anhydrotetracycline) binds to TetR and inhibits its operation.
References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). 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 Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 References (unparsed) here: Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999). 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 Res. 1997 Mar 15;25(6):1203-10. PMID: 9092630 BBa_C0040 TetR Protein is based on the TetR sequence from Elowitz's repressilator. It has been modified to include a rapid degradation LVA tail, and includes the BioBrick standard assembly head and tail restriction sites. The RBS has been removed. The stop codon has been changed from TAA to a double stop codon TAATAA.
June Rhee, Connie Tao, Ty Thomson, Louis Waldman.
One of two requisite genes required for the biosynthesis of phycocyanobilin from heme.
pcyA converts biliverdin IXalpha (BV) to phycocyanobilin (PCB). Strong RBS (efficiency=0.6).
phycocyanobilin:ferredoxin oxidoreductase (PcyA) from synechocystis
Second of two required phycocyanobilin biosynthetic genes.
Conversion of the iGEM parts registry to SBOL2.1