Browsing by Author "Jormanainen, Iina"

The spread of antibiotic resistance in bacteria is a global problem. Horizontal gene transfer (HGT) is the main mechanism implicated in the spread of antibiotic resistance genes (ARG). This study is related to a doctoral thesis project that studies HGT in wastewater microbial community by conducting a microcosm experiment that uses Emulsion, Paired-Isolation and Concatenation PCR (epicPCR) to monitor the spread of ARGs between species. The aim of this study was to introduce synthetic epicPCR primer binding sites inside various ARGs and to test the function of the encoded proteins. The goal was to maintain sufficient protein function, i.e., antibiotic resistance despite the modifications, which allows the further use of modified ARGs in microcosm experiment. The ARGs selected for modifications were dfrB2, ermB, ermC, sul1 and sul2. Sequence-based prediction method was applied to find regions that tolerate insertions inside the proteins encoded by ARGs. The modified ARGs carried in plasmid pUC19 were introduced to Escherichia coli DH5α, which was used as the host in antibiotic susceptibility testing. Antimicrobial gradient method was used to test the antibiotic susceptibility of the strains and to verify the function of the proteins. Six ARGs modified in this study encoded for functional proteins that conferred antibiotic resistance while three modified ARGs did not. Two out of four proteins with insertions in predicted permissive stretches in the middle of a protein maintained their function. The six functional, antibiotic resistance conferring genes designed in this study can be used in further studies utilizing epicPCR. Based on the results of this study, sequence-based prediction method for finding permissive stretches seems useful, but it does not guarantee that the protein function is maintained.