Barkai-Leibler design experiment Part A (Lambda cI) wth cooperativity
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40.
This forms the activator of a Barkai-Leibler oscillator. Differs from BBa_I12000 in that this part includes an extra 434 OR region downstream of the -10 site to allow the repression to take advantage of cooperativity.
This part to work in conjunction with BBa_I12002. This part creates one of the proteins involved in the Modified Barkai-Leibler relaxation oscillator design (activator protein: lambda cI). In the presence of Lambda cI, more Lambda cI will be produced (positively autoregulated). However, if there is 434 cI repressor present in the system, the production of Lambda cI should be repressed. This was implemented in the subpart BBa_I12030, which represents the Operating Regions for the promoter. The fact that the OR-3 site was replaced with the OR-1 site of 434 cI, and that the OR-2 site of 434 cI is included directly after the -10 site should provide enough cooperativity/binding affinity for the lambda cI to be sufficiently repressed when there is 434 cI binding in.
Modified lamdba Prm promoter (repressed by 434 cI with cooperativity) RBS+
Lamdba Prm promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI) with both activation and repression taking advantage of cooperativity.
The O-R1 region of 434 contained 14 base pairs as opposed to the 17 base pairs of the O-R3 site of lambda. Also, it was noticed that the O-R3 site of the lambda included part of the -10 site. Hence, to preserve the spacing and the -10 site, the three nucleotides that were in both the -10 site and the lambda O-R3 site were retained. The 14 nucleotides that were in the O-R3 site and not in the -10 site were replaced with the O-R1 site of the 434. To allow 434 cI to take advantage of cooperativity and to maintain spacing, the -10 site was left undisturbed, but immediately following, the space was replaced with the OR-2 site of 434 cI. Thus, if 434 cI is present in the system, it will bind to both the OR-1 and OR-2 sites of 434, preventing transcription. However, if there is no 434 cI present, then a portion of the OR-2 site will be included in the mRNA, which should not result in any problems.
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
Double terminator consisting of BBa_B0010 and BBa_B0012
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.
Conversion of the iGEM parts registry to SBOL2.1