A Kill Switch with "memory" time repressed by IPTG
Name Number Description CI coding part BBa-C0051 Coding cI Promoter 1 BBa-J23116 Weak constitutive promoter Promoter 2 BBa-R0010 LacI repressible promoter Promoter 3 BBa-R0051 CI repressible promoter Table 1 Biobricks used in the design
We plan to establish a new E.Coli strain without lacI and MazEF system gene to avoid the inference of cell itself. We will test cI background expression level to see if we need to knock out cI. As is shown in Fig.1, Fig.2, Fig.3 and Fig.4, in the ???Stable State???, before poured into the soil, the E. Coli fertilizer is cultured in the medium with IPTG. Promoter 1 is a weak constitutive promoter, so lacI is transcribed and translated at a considerate low level. Therefore, high concentration of IPTG, which binds LacI and changes LacI???s conformation, is able to inactive LacI and open the promoter 2. Then, CI can be highly expressed to repress promoter3. Finally, mazF is inhibited. After fertilizing, no more IPTG exists in the soil to bind LacI. As a result, LacI with a tag which stabilize the protein takes time to accumulate to a certain high concentration, which is the repression threshold of promoter 2, aiming to represses promoter 2 as well as the expression of cI. The time needed to finish this progress is the designed ???memory time???. During this process, E. coli takes its own responsibility to deliver Mo. And after that, it can be killed to reduce the pollution to environment. As is shown in table 1, all the biobricks in the design are from the top 10 most used parts of iGEM to guarantee the feasibility. Name Number Description CI coding part BBa-C0051 Coding cI Promoter 1 BBa-J23116 Weak constitutive promoter Promoter 2 BBa-R0010 LacI repressible promoter Promoter 3 BBa-R0051 CI repressible promoter Table 1 Biobricks used in the design In the future, we plan to model the ???memory time??? and do more wet-experiments to confirm its feasibility. As for the modeling, we will test the expression level of promoter 1, the minimal stand of LacI to repress promoter 2 and the degradation speed of LacI. On the other hand, we will test the background expression of CI after removing IPTG to check if the CI concentration is low enough to open the promoter 3. Then we will strive to make this system more effective.
The Plac insert was PCR'd from the MG1655 strain of E.coli K12.
Inverting regulatory region controlled by LacI (<bb_part>BBa_C0010</bb_part>, <bb_part>BBa_C0011</bb_part>, etc.) The pLac regulatory region is a 243 base-pair sequence with standard BioBrick prefix and suffix sections on its ends. It contains two protein binding sites: CAP, which is generally present in E.coli and is assocciated with cell health and availability of glucose., and LacI, the Lac inhibitor <bb_part>BBa_C0010</bb_part> which binds in an dimerized cooperative manner to inhibit the transcription of the protein that follows. In the presence of lactose or IPTG, an analog of lactose, LacI is unable to correctly bind and inhibit transcription. This allows <bb_part>BBa_R0010</bb_part> to be used as a inverter or as a detector of lactose or IPTG.
LacI binds to this regulator. This part is incompatible with species containing active LacI coding regions. Lactose and IPTG disable the operation of LacI and this regulator. This part is incompatible with environments containing lactose or lactose analogs.
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.
Double terminator consisting of BBa_B0010 and BBa_B0012
Encodes a stable non-specific ribonuclease in ''Bacillus Subtilis''. It is used in conjungtion with ''mazE'' in ''Bacillus Subtilis'' to provide a toxin-antitoxin in various stressful conditions. When transcribtion of both genes is turned off (as both are under contol of same promoter) ''mazE'' will be degraded faster than ''mazF''. There is then no inhibiton of ''mazF'', killing the cell.
Sequence isolated based upon the work of Pellegrini, O., Mathy, N., Gogos, A., Shapiro, L., and Condon, C. (2005) The ''Bacillus subtilis'' ydcDE operon encodes an endoribonuclease of the MazF/PemK family and its inhibitor. ''Mol Microbiol'' 56: 1139???1148.
>gnl|ECOLI|EG10525 lacI "PD00763" (complement(366734..365652)) Escherichia coli K-12 substr. MG1655 http://ecocyc.org/ECOLI/sequence-rc?type=GENE&object=EG10525
Coding region for the LacI protein. LacI binds to the lac promoter BBa_R0010 and inhibits transcription. IPTG binds LacI and inhibits its function, and thus promotes transcription from the lac promoter.
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