Team:TU Darmstadt/Notebook/sec1/K1602038
K1602038 - B0034-enhanced yellow fluorescent protein-SH3-ligand (B0034-eYFP-SH3lig)
The gene eYFP encoding for the enhanced yellow fluorescent protein was provided by the iGEM Registry. Competent E. coli cells of strain Top 10 were transformed via heat shock with the vector pSB1C3 harboring the coding sequence. Colonies were inoculated and the plasmids were extracted. The RBS was added by cloning the construct into the vector pSB1A2 containing the RBS sequence. Therefore, the construct was digested with XbaI and PstI and ligated into the vector, which was digested with SpeI and PstI. Competent E. coli Top 10 were transformed via heat shock. Positive colonies were determined by colony PCR (Figure 1) with VF2 and VR and inoculated, the plasmids were extracted. To connect the enzyme to the in vitro scaffold a linker was added. Therefor, the construct B0034-YFP was used as template for a PCR with VF2 and SH3lig_opt_rev. The PCR product was verified by gel electrophoresis.
Figure 1 Colony PCR of B0034-YFP (1). The size of the amplified product was around 1.1 kbp. DNA marker: 2-Log DNA Ladder. (NEB).