Plasmid construction and transfection

We conduct experiments to building a system using gRNA designed by PAMConAligner. As a model for antibiotic-resistant bacteria, we use a plasmid carrying the AmpR gene. This time, we selected and used BBa_J435300 from the iGEM 2023 kit plate. We determined the gRNA sequence targeting the AmpR gene in BBa_J43530 at PAMConAligner and constructed the gRNA.We also construct a Cas9 plasmid with a gRNA transfected part. This part can be cut with restriction enzyme Bbs I. First, we constructed a Cas9 plasmid with mCherry inserted in this part. This Cas9 plasmid has CmR and it is designed to be expressed only in the presence of IPTG.
(From here, we will refer to the plasmid with mCherry inserted as 'mCherryCas Plasmid' and the plasmid with gRNA inserted as 'gRNACas Plasmid'.)

We introduced gRNA targeting BBa_J435300 into the mCherry part of the constructed Cas9 plasmid by Golden Gate assembly, and we constructed a functional Cas9 plasmid. We performed transformation with the assembly product to obtain E. coli that had acquired the gRNACas plasmid. However, most of the red colonies expressed mCherry. From this, we considered that the efficiency of gRNA introduction into the Cas plasmid was low and that the mCherryCas plasmid remained unchanged. Therefore, we increased the amount of gRNA used in the Golden Gate method and extended the time for digestion and ligation. As a result, the percentage of mCherry-expressing colonies decreased more than before. From this plate, white E. coli colonies that had acquired the gRNA Cas plasmid were selected to create a competent cell with the gRNACas plasmid. The CaCl2 method was used to create this competent cell. Transformation of BBa_J435300 was performed in this competent cell by the heat shock method and cultured on plates supplemented with Amp, Cm, and IPTG. As a result, colonies could be confirmed, and E. coli that acquired both gRNACas plasmid and BBa_J435300 plasmid were obtained.

Figure1. general view of result