Modular Genetic Assembly in E. coli

Native genetic parts were amplified from plasmids using Q5 High-Fidelity DNA Polymerase (New England Biolabs). Where necessary, native genetic parts were made compatible, (i.e., BsaI and BpiI sites were removed) using specific primers. Golden Gate assembly reactions were performed with restriction enzymes BsaI (Thermo Fisher Scientific) or BpiI (Thermo Fisher Scientific), and T4 DNAligase (Thermo Fisher Scientific) according to the protocol of the MoClo Toolkit (Addgene kit # 1000000044). Vectors were transformed into chemically competent Escherichia coli XL-1 blue (Agilent) as per the manufacturer’s instructions. Transformed cultures were grown at 37°C on LB medium with appropriate antibiotic selection for levels 0 and 1 vectors from the MoClo Toolkit (Addgene kit # 1000000044) which are respectively destination vectors for single genetic element and assembled transcription unit as outlined in (Weber et al., 2011 (link)). Four final vectors were constructed where uidA and NAT genes, separated by a 2A peptide, are under control of the Phaeodactylum pFcpB promoter/pFcpA terminator.