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The Final Construct

The Improved Flip Flop (Part: BBa_K510019 and Part: BBa_K510036) is based on the basic flip flop (Part: BBa_K177038) and was built with devices which are in the Registry of Standard Biological Parts.

Here we show the final structure of our improved bistable. As well as the two operons with their protein fusions and the promoter with the asRNA, we show the different restriction sites we have introduced and the enzymes that target each site. Finally, the total length of our construction is indicated in the figure. As for the different sequences used in our construction, here it is a brief description of each one:

1. 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).

2. 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.

3. RBS.3 medium (Part: BBa_B0032): This part is a weak RBS based on Ron Weiss thesis.

4. LacI: Our LacI was taken from E. coli strain K-12 substrain MG1655 chromosome genome and it has a total of 1083 bp.

5. GFP (Part: BBa_E0040): Green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP.

6. DAS+4: Short tag at the end of the proteins. For more information see Figure 2 of Proteolysis section.

7. Temperature sensitive cI (Part: BBa_K177050): Temperature-sensitive version of the lambda phage repressor. Functions in 30°C and it's inactivated in 42°C

8. 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.

9. 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.

10. 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.

11. RybB asRNA: Taken from Bouvier et al.(2008). For further information see Figure 2 of the asRNA section.