Team:Guelph/Results

Results

The cloning was first accomplished using E. coli as the organism. The guide RNAs were cloned into the pHSN6A01 plasmid, creating six different plasmids when including the multiplex. Once the E.coli transformation was complete and isolated colonies were identified, the plasmids were confirmed through a PCR Verification protocol using Froggabio and colony water. The banding size expected for a positive test is approximately 20 base pairs, which is depicted in Figure 1. Upon confirmation, glycerol stocks were made in order to properly preserve the plasmids and the agrobacterium transformation started. The agrobacterium transformants were confirmed in a similar way as their E. coli counterpart, which is depicted in Figure 2. Once the agrobacterium plasmids were created, the AlcR promoter-CRISPRa plasmid was created using restriction enzymes HindIII and EcoRI, this was confirmed using a PCR verification and gel electrophoresis depicted in Figure 3.