Transposon Mutagenesis in Shewanella Oneidensis
Mentor 1
Daad Saffarini
Location
Union Wisconsin Room
Start Date
27-4-2018 1:00 PM
Description
The cAMP receptor protein CRP is involved in controlling the expression of aerobic reductase genes. Adenyl cyclases synthesize cAMP in response to environmental stimuli and aid in activation of CRP. Shewanella oneidensis mutants that have a deficiency in CRP production show no growth under anaerobic condtions. These mutants are able to grow aerobically, but to a lesser extent than wild type. In addition, cAMP levels are also regulated by the activity of a phosphodiesterase cpdA (SO_3901). When deleted, SO_3901 doesn’t affect anaerobic growth. It does however, cause a greater deficiency in aerobic growth. The purpose of my work is to investigate the reason for the difference in aerobic growth deficiency between CRP and SO_3901 mutants. SO_3901 suppressor mutants have been generated through transposon mutagenesis to identify potential factors that may contribute to the loss of aerobic respiration in SO_3901. The plasmid pminiHIMAR RB1 was transferred into SO_3901 from E. coli WM3064 by conjugation. Suppressor mutants were selected on basal media containing lactate and kanamycin. Mutant chromosomal DNA was isolated and digested using the restriction enzyme BamHI. The fragments were ligated together and used to transform E. coli. The resulting plasmids were used for sequencing. This research is ongoing and isolated suppressor mutants will continue to be tested for aerobic growth in basal media. The mutants will also be complemented to determine the effect on aerobic growth. Finally, additional suppressor mutants will be generated and their sites of transposon insertion will be identified.
Transposon Mutagenesis in Shewanella Oneidensis
Union Wisconsin Room
The cAMP receptor protein CRP is involved in controlling the expression of aerobic reductase genes. Adenyl cyclases synthesize cAMP in response to environmental stimuli and aid in activation of CRP. Shewanella oneidensis mutants that have a deficiency in CRP production show no growth under anaerobic condtions. These mutants are able to grow aerobically, but to a lesser extent than wild type. In addition, cAMP levels are also regulated by the activity of a phosphodiesterase cpdA (SO_3901). When deleted, SO_3901 doesn’t affect anaerobic growth. It does however, cause a greater deficiency in aerobic growth. The purpose of my work is to investigate the reason for the difference in aerobic growth deficiency between CRP and SO_3901 mutants. SO_3901 suppressor mutants have been generated through transposon mutagenesis to identify potential factors that may contribute to the loss of aerobic respiration in SO_3901. The plasmid pminiHIMAR RB1 was transferred into SO_3901 from E. coli WM3064 by conjugation. Suppressor mutants were selected on basal media containing lactate and kanamycin. Mutant chromosomal DNA was isolated and digested using the restriction enzyme BamHI. The fragments were ligated together and used to transform E. coli. The resulting plasmids were used for sequencing. This research is ongoing and isolated suppressor mutants will continue to be tested for aerobic growth in basal media. The mutants will also be complemented to determine the effect on aerobic growth. Finally, additional suppressor mutants will be generated and their sites of transposon insertion will be identified.
