Team:TU Darmstadt/Notebook/sec1/K1602039
K1602039 - B0034-enhanced yellow fluorescent protein-PDZ-Ligand (B0034-eYFP-PDZlig)
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 using a PCR with the oligonucleotides VF2 and YFP_fus_rev. The PCR product was verified by gel electrophoresis, digested with DpnI and purified. 1 ng of this product was used to add a ligand to the linker. Therefor, a PCR with oligonucleotides VF2 and PDZlig_opt_rev was used (Figure 2). The product was digested with EcoRI and PstI, cloned into pSB1C3 and transformed into competent E. coli Top 10. Colonies were screened by colony PCR using VF2 and VR (Figure 3).
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).
Figure 2 Colony PCR of B0034-YFP-Linker (1). The size of the amplified product was around 1.0 kbp. DNA marker: 2-Log DNA Ladder. (NEB).
Figure 3 Colony PCR of B0034-YFP-Linker in pSB1C3 (2+3). The size of the amplified product was around 1.0 kbp. DNA marker: Gene Ruler 1 kB DNA Ladder. (NEB).