Team:Hong Kong-CUHK/Improve
Design improvement
The diffusible signal factor (DSF) sensing system, developed by the previous team (Dundee, 2014), utilizes the manA promotor in their reporter construct. Their report recorded that their system shows visible leakage of the green fluorescent signal even without the upstream sensing component RpfC, RpfG and Clp.
We reconstruct the whole detection system to bypass the leakage manA promotor by utilizing the pspA promotor (BBa_K3071013), Clp-binding upstream sequence (BBa_K3071011 and BBa_K3071012) and PspF TAD (BBa_K3071007). We constructed a synthetic promotor CBSI & II regulated pspA promotor (BBa_K3071014) that shows no visible signal leakage. Our rt-qPCR data in azathioprine drug assay and DSF assay also indicate that the construct is a functional construct.
Besides tackling the signal leakage of the original design, we also redesigned the reporter construct signal outcome. We replaced the signal outcome from Green fluorescence protein GFP to eforRED Red chromoprotein (BBa_K592012). The advantage of chromoprotein is that their color could be visible without the need for excitation, making the design less laboratory-dependent and allowing naked-eye detection.
Codon optimization and Enhancement of protein expression
All our foreign protein encoding sequence is being codon optimized to suit for the Escherichia coli expression including RpfC (BBa_K3071000), RpfG (BBa_K3071002) and Clp (BBa_K3071004).
BBa_K1315002: It found to have multiple stop codon at the position 42, 69, 251, 453, 715 in amino acid sequence. We also find that the sequence is actually translated in the lower strand in 5’→3’ direction and recorded as the new biobrick (BBa_K3071000)
BBa_K1315003: Our western blot result indicate this protein encoding sequence is not optimized to be expressed inside E. coli. Our new biobrick (BBa_K3071002) shows a much higher expression rate than the old biobrick, which confirm the success of codon optimization. Please refer to the Wet lab: Result figure 7.
BBa_K1315005: Sequence optimized and recorded as new biobrick (BBa_K3071004)