Part:BBa_K2083009

Coding region for a constitutively expressed GFP reporter. A section of the GAPDH gene is used as the 5’ UTR, as it is a known successful target site for Cas9.

Figure 1: Wild Type ATG GFP Reporter Fluorescence

Figure 2: Mutated Start Codon GFP Reporter Fluorescence

The data indicates relatively high efficiency in the ATG/ACG reporters. According to the microscopy data the ATG reporter showed 70.57% of fluorescence compared to the positive control, while the ACG reporter only gave 4.21%. Therefore, the mutated start codon is proven to be able to produce the difference between the two versions of the reporter.

Figure 3: Efficiency of ATG/ACG reporters compared to positive control. Error bars represent standard error between normalized results from 3 biological replicates of the experiment.

Figure 4: ATG/ACG reporters efficiency confirmation by flow cytometry. The difference between the wild type and mutant reporter is consistent with the microscopy data.

For how the values were calculated see this: https://static.igem.org/mediawiki/2016/c/c7/T--WPI_Worcester--Imaging_Analysis.pdf

For the fluorescence values see here:

https://static.igem.org/mediawiki/2016/d/d2/T--WPI_Worcester--ATG_Trial1.pdf: Trial 1

https://static.igem.org/mediawiki/2016/1/1a/T--WPI_Worcester--ATG_Trial2.pdf: Trial 2

https://static.igem.org/mediawiki/2016/8/81/T--WPI_Worcester--ATG_Trial3.pdf: Trial 3