Team:TU Darmstadt/Notebook/sec1/K1602034
K1602034 - Acetyl Esterase-SH3-Ligand (aes-SH3lig)
The from the iGEM registry received vector pSB1C3 harboring the sequence for aes with natural RBS was transformed into competent E. coli strain Top10 cells via heat shock. Colonies were inoculated and 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 aes_fus_rev. The PCR product was verified by gel electrophoresis (Figure 1), digested with DpnI and purified. 1 ng of this product was used to add a ligand to the linker. Therefor, oligonucleotides VF2 and SH3lig_opt_rev were used. The sample was loaded on an agarose gel for further gel extraction (Figure 2). Afterwards, the gel-extracted product was digested with EcoRI and PstI, cloned into pSB1C3 and transformed into competent E. coli Top 10 cells. Colonies were analyzed by colony PCR using oligonucleotides VF2 and VR (Figure 3).
Figure 1 Linker-PCR of B0034-aes (1). The size of the amplified product was around 1.0 kbp. DNA marker: Gene Ruler 1 kB DNA Ladder. (NEB).
Figure 2 PCR of SH3 domain added to B0034-aes-Linker (1). The size of the amplified product was around 1.3 kbp. DNA marker: Gene Ruler 1 kB DNA Ladder. (NEB).
Figure 3 Colony PCR of B0034-aes-Linker-SH3 in pSB1C3 (1-6). The size of the amplified product was around 1.3 kbp. DNA marker: Gene Ruler 1 kB DNA Ladder. (NEB).