BL oscillator, cooperativity + replaced -10 region (Llac), reporter protein, kickstart
This part is a Barkai-Leibler oscillator with cooperativity for the repressor (two 434 cI OR site). A reporter protein (EYFP) and a kickstart is included. This part differs from BBa_I12045 in that the -10 site for the promoter region of the Lambda cI has been replaced with the Lac -10 region.
There is cooperativity for 434 cI in this oscillator. A protein EYFP (with degradation tag) coding region is placed directly after the lambda cI coding region in order to keep track of the lambda cI levels. This part also includes a "kickstart" that will yield lambda cI if ATC is added. (Differs from BBa_I12045 due to the swapped -10 region of the Lambda cI (LLac))
Barkai-Leibler design experiment part B (434cI)
This forms the repressor of a Barkai-Leibler oscillator. The 434 cI has no LVA tag (BBa_I12014 is the same as BBa_C0052 with no LVA tag).
This part to work in conjunction with BBa_I12000. This part creates one of the proteins involved in the Modified Barkai-Leibler relaxation oscillator design (repressor protein: 434 cI). In the presence of Lambda cI, this part will create 434 cI, which will in turn repress the production of Lambda cI in BBa_12000. The LVA tag was removed from the 434 cI coding region due to the fact that we want the repressor to be as potent as possible, thus, if it doesn't degrade as quickly, the relative strength should be increased.
Kickstart for the Barkai-Leibler oscillator
This part is designed for "kickstarting" the Barkai-Leibler oscillator by producing lambda cI.
Addition of ATC will repress the tetR and this will result in production of lambda cI.
Activator for BL oscillator with reporter protein, (cooperativity and L-strain -10 region)
This part forms the activator part of the Barkai-Leibler oscillator. It has cooperativity for the repressor protein 434 cI (two OR sites). It includes a reporter protein. This part differs from BBa_I12044 in that the -10 site for the promoter region of the Lambda cI has been replaced with the Lac -10 region.
This part has cooperativity for the repressor 434 cI (two 434 OR sites). Also, there is a possibility that this Lambda cI promoter region may be more powerful than that of BBa_I12044 since the -10 region has been replaced with that of LLac. A reporter protein (EYFP) coding region is placed after the lambda cI coding region. This will make it possible to keep track of the lambda cI levels in the cell.
Nikolnikov,S., Posfai,G. and Sain,B. "The construction of a versatile plasmid vector that allows direct selection of fragments cloned into six unique sites of the cI gene of coliphage 434"
The 434 cI repressor protein coding sequence is a 710 base-pair sequence with the standard RBS-compatible BioBrick prefix and the standard BioBrick suffix sections on its ends. It binds to the 434 regulatory sequence, BBa_R0052. The sequence has no LVA tag nor RBS.
Essentially the same as BBa_C0052 but with the LVA tag removed.
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.
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
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
Double terminator consisting of BBa_B0010 and BBa_B0012
Modified lambda Prm promoter (OR-3 obliterated)
Lambda Prm promoter modified to be activated but not repressed by the lambda repressor (cI)
In wild type lambda phage, the OR3 site (-27 to -11) reads "tatcccttgcggtgata" on the sense strand of DNA. To prevent lambda repressor (cI) from binding to this site, the 4th through 10th nt of OR3 were replaced by the 7 nt between OR2 and OR1, "aaatagt" (-57 to -51), which in effect mutates 5 nt of the promoter. These nt were chosen to be about halfway between the -10 and -35 boxes of the promoter. Furthermore, since these nt already act as a spacer in wild-type phage, it is hoped they will not create undesired interactions.
Modified lambda P(RM) promoter: -10 region from P(L) and cooperatively repressed by 434 cI
Bushman, F. D. The bacteriophage 434 right operator roles of O-R1, O-R2, and O-R3. J. Mol. Biol. (1993) 230, 28-40. ; 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 Research, 1997, Vol.25, No.6 (1203-1210)
Lamdba P(RM) promoter modified to be activated by lamda repressor (cI) and repressed by 434 repressor (cI). Two operator sites are used for both activation and repression to enhance cooperativity. Additionally, the -10 region from lambda P(L) was used instead of that from P(RM).
The O-R3 site of lambda (17 nt) was replaced by the O-R1 site of 434 (14 nt) to facilitate repression by 434 cI. The new 434 O-R1 is 5'-justified with the lambda O-R3 it replaced, leaving the -10 sequence intact. The 434 O-R2 site replaces the wildtype labmda promoter starting at -5, thereby preserving the 8 nt between 434 O-R1 and O-R2 in the wildtype. The mRNA transcript will contain an additional 9 nt compared to wildtype lambda phage. This part differs from BBa_I12036 because the -10 region from P(L) was used instead of that from P(RM).
enhanced yellow fluorescent protein derived from A. victoria GFP
Modified from <bb_part>BBa_E0031</bb_part>
Yellow fluorescent protein (EYFP) reporter coding sequence without the Ribosome Binding Site. Modified with an LVA tail for rapid degradation of the protein and faster fall time for the emission. Annotated mutation cause a Q81L mutation that appears to not be near the active site.
BBa_E0032 yellow fluorescent protein is based on BioBrick part BBa_E0031. 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.
Conversion of the iGEM parts registry to SBOL2.1