Part:BBa_K510019:Design
Improved Flip Flop (Module I)
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3130
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 940
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 4392
Illegal AgeI site found at 4504 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 271
Design Notes
As for the different sequences used in our construction, here it is a brief description of each one:
- cI promoter (Part: BBa_R0051): The cI regulated promoter is based on the pR promoter from bacteriophage lambda. The promoter has two DNA binding sites for lambda cI repressor BBa_C0051. 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).
- Lac promoter (Part: BBa_R0010): This part is an inverting regulator sensitive to LacI and CAP. It contains two protein binding sites. The first binds CAP protein, which is generally present in E.coli and is associated with cell health and availability of glucose. The second one binds LacI protein.
- RBS.3 (medium) (Part:BBa_B0032): This part is a weak RBS based on Ron Weiss thesis.
- LacI: Our LacI was taken from E. coli strain K-12 substrain MG1655 chromosome genome and it has a total of 1083 bp.
- GFP (Part: BBa_E0040): Green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP.
- DAS+4: Short tag at the end of the proteins. For more information see Figure 2 of Proteolysis section.
- Temperature sensitive cI (Part: BBa_K177050): Thermosensitive version of the lambda phage repressor. Functions in 30°C and it's inactivated in 42°C
- SspB: Taken from McGinness et al. (2006). For more information, see Proteolysis section. The OmpN::SspB fusion is detailed in Figure 5 of the asRNA section.
- RFP (Part: BBa_E1010): Highly engineered mutant of red fluorescent protein from Discosoma striata (coral). Monomeric RFP with its excitation peak at 584 nm and its emission peak at 607 nm. The OmpN::RFP fusion is detailed in Figure 5 of the asRNA section.
- Double terminator (Part BBa_B0015): Double terminator consisting of BBa_B0010 and BBa_B0012.This is the most commonly used terminator and it seems to be reliable.
Source
It's a composite part which comes from different genes forming the bassic flip-flop with anothers improvements. According to Design Notes, the main source was the Registry of Standard Biological Parts although also nbci database was used to the following parts: LacI and SspB. Besides Tag sequences (DAS4, asRNA and OmpN fusion) were obtained from each articles (see references). Finally, the complete construction was synthesized commercially.
References
- Timothy S. G., Charles R. C. & James J. C. (2000). Construction of a genetic toggle switch in Escherichia coli. Nature 403, 339-342.
- McGinness, K.E., Baker, T.A., and Sauer, R.T. (2006). Engineering Controllable Protein Degradation. Molecular Cell 22, 701-707.
- Bouvier, M., Sharma, C.M., Mika, F., Nierhaus, K.H., and Vogel, J. (2008). Small RNA Binding to 5’ mRNA Coding Region Inhibits Translational Initiation. Molecular Cell 32, 827-837.
For further information please vistit our [http://2011.igem.org/Team:UPO-Sevilla wiki] or contact with us at igem@upo.es