After creating Bba_K1497001, we added a strong RBS (Bba_B0034) in front of the coding part of CHS. For this reason we used PCR with primers coding for RBS, Biobrick prefix and Biobrick suffix (RBS_CHS_pre and CHS_psb1c3_Suf2). We analyzed the PCR via agarose gel electrophoresis and cleaned up positive samples with DpnI digestion and Wizard PCR Clean Up Kit (Promega).  We cut the cleaned up samples with EcoRI and PstI and ligated them with a previously digestion of mRFP-pSB1C3 with EcoRI + PstI. We transformed the ligation in Top10 E. coli via heat shock method and spread out on CMP-LB agar plates. After incubating overnight, we analyzed obtained colonies via colony-PCR and agarose gel electrophoresis. Positive colonies were inoculated in 5 ml LB with CMP and plasmids were isolated after incubating for 16 h at 37 °C.

For further verification we send the samples to Eurofins for sequencing.