Team:Jilin China/Improve

IMPROVE

Improve

  • Improvement

    This year, we choose Golden Gate Assembly especially for construction. (Learn about our Construction? See our Construction Page) BsaI and BbsI are used as Type IIs restriction endonucleases in our construction device. At the same time, we decided to use sfGFP as our reporter protein due to its faster folded feature and higher fluorescence intensity. However, sfGFP in iGEM part registry (BBa_I746916) has a BbsI recognition site, which could be affected during GoldenGate Assembly. Hence, first coming into our mind was to do a site mutagenesis, BbsI recognition site GAAGAC was altered into GAGGAT while amino acids sequence kept the same. Then we got our sfGFP(BbsI free) part(BBa_K2541401) , a BbsI-free sfGFP and especially for Golden Gate Assembly(Fig 1.).

    Figure 1. Site mutagenesis for sfGFP BbsI free.

    Because we cannot guarantee the mutated sfGFP has the same fluorescence expression as the previous one, after further study, we found a codon optimized sfGFP[1,2]without BbsI recognition site.

    The free of the BbsI cleavage site was confirmed by nucleic acid electrophoresis(Fig.2).

    Figure 2. L1: 1kb DNA marker; L2: BBa_I746916; L3: BBa_I746916+BbsI; L4: BBa_K2541401; L5: BBa_K2541401+BbsI; L6: BBa_K2541400; L7: BBa_K2541400+BbsI; L8: 1kb DNA marker.

    Next, we got three types of sfGFP emission and excitation spectra.(Fig.3)

    Figure 3. Three types of sfGFP emission and excitation spectra

    For further characterization, we detected the expression intensity of these three types of sfGFP. According to the results(Fig.4), we found out that the expression intensity of sfGFP_optimism is nearly 2.6 as much as the other two types. As a result, we finally choose sfGFP_optimism as our reporter protein.

    Figure 4. Three Types of sfGFP expression in E.coli.

    More importantly, due to its standardization in Golden Gate Assembly and better property, part sfGFP_optimism(BBa_K2541400) is greatly competitive in Best Basic Part.

  • Reference

    • [1]Segall-Shapiro, Thomas H., Eduardo D. Sontag, and Christopher A. Voigt. "Engineered promoters enable constant gene expression at any copy number in bacteria." Nature biotechnology 36.4 (2018): 352.
    • [2]Overkamp, Wout, et al. "Benchmarking various GFP variants in Bacillus subtilis, Streptococcus pneumoniae and Lactococcus lactis for live cell imaging." Applied and Environmental Microbiology (2013): AEM-02033.