Team:Tianjin/Improved Part
mRFP reporter with weak constitutive promoter
(BBa_K3112050)
Compared with the original part: BBa_J04450, we replace the lac1 promoter with a member of a constituent promoter family (BBa_J23115)which has a weak expression intensity but can still be clearly observed under fluorescence microscopy.
We hope that this part can be used in co-expression with some colored products, and its weak expression intensity will not affect the observation of its colored products.
Cloning
Figure 1. iGEM Tianjin Part construction
Two restriction sites EcoRI and BamHI were added to both ends of the whole part by PCR. It is for the convenience of other teams to carry out further cloning operations or add other constitutive promoters or terminators.
After digestion with the corresponding enzyme, T4 ligase was used to connect the two segments to plasmid pRS416.
Figure 2. Plasmid profiles
Figure 3. Color contrast of bacterial solution (left: mRFP with J23110P，medium: mRFP with J23115P，right: Original part)
Figure 4. Phenotypic analysis (Up:original part；down:improved part)
Figure 5. Contrast of fluorescence intensity before and after improvement
It has been proved that the phenomenon that the original expression of our improved part is too high has been eliminated, and the expression of the improved part has been reduced a lot. In this way, when some colored products are expressed , mRFP itself will not affect the observation of the products.
