Induction Of Apoptosis In Escherichia Coli Cells

Victoria McCarthy

School Name

South Carolina Governor's School for Science and Mathematics

Grade Level

12th Grade

Presentation Topic

Microbiology

Presentation Type

Mentored

Mentor

Mentor: Tzuen-Rong Tzeng, Department of Biological Sciences, Clemson University

Abstract

Bacterial cells have been proven to go through programed cell death similar to the apoptosis of mammalian cells. MazF is a bacterial toxin that plays an important role in bacterial apoptosis by cleaving mRNA when it is activated. MazE is the antitoxin that prevents MazF from being activated in the lag and log phases of bacterial cell growth. MazF is capable of cleaving mRNA at the ACA sequence, but the toxin is only active in the stationary phase when there are not enough nutrients to sustain the population. mazF is able to cleave itself due to the ACA sequence on its mRNA, which reduces apoptosis of the bacterial cells. The aim of this research is to study the effect of altering the ACA to another sequence encoding the same amino acid and study its role in bacterial apoptosis. To achieve this goal, mazF was amplified using PCR and digested with XhoI and HindIII. The digested product was cloned into the pBAD vector under the control of an arabinose-inducible promoter E. coliDH10B and transformed with pBAD carrying the modified mazF. The transformed E. coli were grown on M9 minimum medium supplemented with 0.2% arabinose, and 100 µl of bacteria were plated onto M9 plates every two hours for 24 hours. The plates were incubated at 37º C and the number of colonies counted after 24 hours of incubation. A 3-log reductions in population in cells expressing the altered mazF mRNA 8 hours post arabinose-induction was observed.

Start Date

4-11-2015 9:30 AM

End Date

4-11-2015 9:45 AM

COinS
 
Apr 11th, 9:30 AM Apr 11th, 9:45 AM

Induction Of Apoptosis In Escherichia Coli Cells

Bacterial cells have been proven to go through programed cell death similar to the apoptosis of mammalian cells. MazF is a bacterial toxin that plays an important role in bacterial apoptosis by cleaving mRNA when it is activated. MazE is the antitoxin that prevents MazF from being activated in the lag and log phases of bacterial cell growth. MazF is capable of cleaving mRNA at the ACA sequence, but the toxin is only active in the stationary phase when there are not enough nutrients to sustain the population. mazF is able to cleave itself due to the ACA sequence on its mRNA, which reduces apoptosis of the bacterial cells. The aim of this research is to study the effect of altering the ACA to another sequence encoding the same amino acid and study its role in bacterial apoptosis. To achieve this goal, mazF was amplified using PCR and digested with XhoI and HindIII. The digested product was cloned into the pBAD vector under the control of an arabinose-inducible promoter E. coliDH10B and transformed with pBAD carrying the modified mazF. The transformed E. coli were grown on M9 minimum medium supplemented with 0.2% arabinose, and 100 µl of bacteria were plated onto M9 plates every two hours for 24 hours. The plates were incubated at 37º C and the number of colonies counted after 24 hours of incubation. A 3-log reductions in population in cells expressing the altered mazF mRNA 8 hours post arabinose-induction was observed.