title: Development of a suicidal vector-cloning system based on butanal susceptibility due to an expression of YqhD aldehyde reductase.
abstract: Previously, we observed butanal/propanal sensitivity of Escherichia coli K-12 when cells overexpress YqhD protein, a NADPH dependent aldehyde reductase, possibly due to an accumulation of butanol/propanol in vivo as the reaction products. Based on this finding, we developed a suicidal vector-cloning system derived from pUC19, in which lacZ was substituted with the yqhD gene. As a result, when foreign DNA was inserted into its multiple cloning sites by disrupting an expression of YqhD, the recombinants survived on butanal/propanal containing plate, whereas cells containing the YqhD vector died because of the alcohol production by YqhD. The cloning efficiency, estimated based on colony PCR and enzyme digestion, was achieved more than 90% when the suicidal vector system was used. Moreover, the plasmid vector itself was stably maintained in the cell, presumably due to its ability to remove toxic aldehydes being accumulated in E. coli cell by metabolic stress.
authors.affiliation: Department of Life Sciences, Korea Advanced Institute of Science and Technology, Daejeon, 305-701, Republic of Korea.
authors.forename: Changhan
authors.initials: C
authors.lastname: Lee
authors.forename: Chankyu
authors.initials: C
authors.lastname: Park
mesh_terms: D000449:Aldehyde Reductase / Q000235:genetics* / Q000378:metabolism; D000440:Butanols / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D048168:Escherichia coli K12 / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000440:Butanols; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; D000449:Aldehyde Reductase; C528732:YqhD protein, E coli