Synthetic biology: Generation of new GFP variants with improved characteristics
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This project aimed to create new fluorescent protein (FPs) variants using the GFPmut3b, EYFP and mRFP1 through fusing two FPs together. The proximity of these fused FPs was expected to result in altered light absorption and fluorescence emission spectra compared to the single FPs. In order to fuse the Fps, plasmids were generated by genetically fusing the coding sequences of two FPs into the expression vector pSB1AK3 with a lacI IPTG-inducible promoter. Gibson cloning and CPEC cloning were used to genetically link the FP. Sequence analysis of the generated constructs, however, indicated that two identical FPs were integrated into the expression vector. Characterization of the linked FPs for light absorption and fluorescence emission showed that their spectral features were identical to the original FPs with absorption maxima at 501 nm, 514 nm, 584 nm and fluorescence emission peaks at 511 nm, 527 nm and 607 nm, for GFPmut3b, EYFP and mRFP1 respectively. The maturation of expressed single FPs and linked FPs was also characterized by inhibiting protein expression with the inhibitor chloramphenicol.