Difference between revisions of "Part:BBa K3257101"

Revision as of 03:02, 18 October 2019

Cre Recombinase with SsrA(WVLAA)-aTc Inducible

For the best composite part, we would like to present you with our Cre recombinase together with degradation tag SsrA(WVLAA)(https://parts.igem.org/Part:BBa_K3257101). Cre recombinase (BBa_K3257044 https://parts.igem.org/Part:BBa_K3257044) is responsible for the recombination process between the reverse-transcribed cDNA and the target Gene of Interest (GOI), which is rather one of the most important processes in our system.

Because of the leakage of uninduced Cre expression, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different types of degradation tags after the CDS of Cre. Using EGFP (BBa_E0040 https://parts.igem.org/Part:BBa_E0040) as a reporter, we can see that the degradation rate of degradation tag with the amino acid residue sequence of AANDENWVLAA (BBa_K3257073 https://parts.igem.org/Part:BBa_K3257073) is moderate for Cre recombinase to reach a steady but relatively low level of expression (Figure 1).

Transformed into BL21 (DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that homologous recombination happens between the same lox sites instead of gene knockout (Figure 2).

Combining these two results, we show that this composite part is able to express Cre recombinase which functions well in an E.coli cell at a relatively low expression level, which is we need for new recombinants accumulating during the R-Evolution process.

In the future, iGEMers and synthetic biologists may use this part for homologous recombination with different degradation tags and promoters based on their own requirements.

Sequence and Features