Part:BBa_K3257101

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). 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, which is rather one of the most important processes in our system.

In order to deal with the leakage of uninduced Cre recombinase expression, which further establishing a more stringent control of the expression of Cre and agree to the modeling results that the concentration of Cre should be at a low level for the recombinant plasmids to accumulate, we have spent much effort in lowering the steady-state expression level of Cre. An important approach is to attach 5 different SsrA 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).

Figure 1. Degradation tag greatly reduces the protein level at stable state. WT represents the positive control of EGFP without any tag attachment. The five degradation tags are represented by their last five amino acid sequence. The vertical axis shows the quantitative analysis of EGFP fluorescence (excitation wavelength: 485 nm; detection wavelength: 528 nm), normalized by cell amount (OD600). The fluorescence is quantified by the concentration of green fluorescein, cell number is quantified by the number of silicon beads, both are from the distributed measurement kit. Fluorescence below detection level is eliminated. Error bar stands for the SEM of 3 replicates. t-test is performed between WT and each degradation tag, P<0.0001 (****).

Transformed into BL21 (DE3) with another plasmid containing lox sites and analyzed by colony PCR, we can see that DNA cleavage between identical-oriented loxP sites happens indicating the normal function of Cre recombinase(Figure 2).

Figure 2. The verification of DNA cleavage between LoxP sites. Plasmids containing the Cre gene were co-transformed with plasmids containing mCherry flanked by LoxP sites. A pair of sequencing primers were used to amplify the mCherry gene. PCR product would be around 1000 bp while fragments cleaved by Cre recombinase would be approximately 250 bp. The positive control group is the PCR product of plasmids containing the original mCherry gene. The negative control group is the PCR product of plasmids containing the Cre gene only.

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 436
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 122
  
• 1000
  COMPATIBLE WITH RFC[1000]