Blue Light Sensing System With GFP And BFP Reporter
CcdB protein is a topoisomerase poision which affects DNA gyrase and inhibits the segregation of the chromosomal DNA.
Blue Light Sensing System with GFP Reporter
This part is a combination of BBa_K592016, BBa_K592006, and BBa_K592010 in that order. We also add a promoter called promoter 104 so that teams can use this part to test the blue light system easily. When the cells are kept in dark, they will express amilGFP, a yellow chromoprotein. This protein causes the cells to have a yellow color. When the cells are exposed to blue light, the amilGFP won't be expressed and the yellow color can't be seen.
How to add the promoter 104 into the part.
PFixK2-lambda C1-B0034-amilCP blue light sensor output
FixK2 promoter: Bradyrhizibium japonicum amilCP: Acropora millepora
FixK2 promoter is the downstream signaling promoter of YF1/FixJ blue light-sensing system. The YF1/FixJ system become "inactive" when illuminated by blue light, and "active" in darkness. Needless to say, a system that works in the opposite way with lights activating and darkness inactivating is more desirable. Therefore, an inverter was placed behind FixK2 promoter. When in dark, YF1 becomes phosphorylated and activates FixJ, its own RR. FixJ in turn binds to FixK2 promoter, allowing the transcription of the lambda C1, thus repressing the amilCP output. In light however, YF1/FixJ has no effect on FixK2 promoter, which in turn has near-zero activity. The lambda C1 concentration drops, eventually amilCP will be produced.
The Q04510 inverter contains B0015 double terminator which gives unspecific primer binding of VR. Touchdown PCR was required to amplify the sequence for screening.
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
Vinay S Mahajan, Voichita D. Marinescu, Brian Chow, Alexander D Wissner-Gross and Peter Carr IAP, 2003.
Ytva comes from the genome of Bacillus subtilis. FixL derives from the genome of Bradyrhizobium japonicum.
YF1 is a fusion protein of YtvA (B.subtilis) and FixL(Br.japonicum). It contains the N-terminal region of YtvA (1-127) and C-terminal region of FixL (258-505), interconnected by a helical linker. This fusion protein is designed by Andreas Moglich and Keith Moffat. As shown in previous research, Illumination of the fusion kinase YF1 reduced net kinase activity by ∼1000-fold in vitro. YF1 also controls gene expression in a light-dependent manner in vivo.
The sequence information was provided in Moglich and Moffat's article. The physical DNA was synthesized by GenScript. Codon-optimization may enhance its expression.
FixJ comes from the genome of Bradyrhizobium japonicum.
FixJ comes from the genome of Bradyrhizobium japonicum. It's the wild-type response regulator to YF1, the blue light-sensing fusion protein.
The sequence was information was provided by Moffat (Moffat et al. 2009). The DNA was synthesized by GenScript.
FixK2 promoter omes from the genome of Bradyrhizobium japonicum.
FixK2 promoter is the wild-type promoter to which phospho-FixJ binds. FixJ in turn can be regulated by YF1, the blue light-sensing protein. This promoter shows very little leaky activity in the absence of FixJ.
The sequence information was provided by Moffat (Moffat et al. 2009). The DNA was synthesized by GenScript.
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).
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.
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.
cI repressor from E. coli phage lambda (+LVA)
Elowitz, M. B. Transport, Assembly, and Dynamics in Systems of Interacting Proteins. Thesis, Princeton Univ., Princeton (1999).
Coding region for the cI repressor based on cI repressor from bacteriophage lambda modified with an LVA tail for rapid degradation of the protein. cI repressor binds to the cI regulator (BBa_R0051).
References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 References (unparsed) here: A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000 Octamerization of CI repressor is needed for effective repression of PRM and efficient switching from lysogeny. Ian B. Dodd,1 Alison J. Perkins, Daniel Tsemitsidis, and J. Barry Egan , Genes and Development (Vol 15, No. 22) 3013-3022: 2001 BBa_C0051 cI repressor is based on the cI repressor from the Elowitz's repressilator. It has been modified to include a rapid degradation LAA 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.
Vinay S Mahajan, Brian Chow, Peter Carr, Voichita Marinescu and Alexander D. Wissner-Gross
A synthetic oscillatory network of transcriptional regulators , Elowitz M.B. , Leibler S., Nature(403),335-38: 2000
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.
Conversion of the iGEM parts registry to SBOL2.1