For best composite part, we would like to present you with our Cre recombinase together with degradation tag. Cre recombinase (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) as a reporter, we can see that the degradation rate of degradation tag with amino acid residual sequence of AANDENWVLAA (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 axe 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 are 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). For more information of other composite parts, please refer to the part collection page and the iGEM Part Registry ranging from BBa_K3257100 to BBa_K3257118.
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.
For more information of other composite parts, please refer to the part collection page and the iGEM Part Registry ranging from BBa_K3257100 to BBa_K3257118.