Survival
Undergraduate Category: Physical and Life Sciences Degree Seeking: B.S. Biochemistry Abstract ID# 1687
Characterization of Cellular Responses to DNA Alkylation in E. coli Jadesola Akanji, Macee Qi, Ariel Aiken, Alexis Hester, Caitlin Kramer, Becky Leifer, Mark Muenter, Penny Beuning
Opportunity
Impact
Prepare 96 Well Plate 1. 180µL 0..85% saline à rows C-‐G 2. 100µL of each time point à row B 3. Serial dilutions of 20µL from row BàC, CàD, DàE…
Chemical Dose Shock 1. 90µL serially dilute BnBr à S eppie 2. S eppies placed on shaker in 37ºC until time points
Plate Subcultures 1. On 50mL LB plate: 5µL of each row from G à B starting from bottom to top 2. Incubate inverted plates in 37ºC for 14-‐16 hours
G-‐C base pair
dam + BnBr
mutH + BnBr
1000
1000
wt + B nBr dam + B nBr
10
1 0
30
60
100
wt + BnBr mutH + BnBr
10
1
90
0
Time (min.)
30
60
% S urvival
% S urvival
100
1000
100
wt + BnBr mutY + BnBr 10
1
90
0
Time (min.)
mfd + BnBr
Data Analysis 1. Count colonies for each time point spot on LB plates 2. Record in lab notebook 3. Graph exposure time vs. % survival
mutY + BnBr
30
60
90
Time (min.)
nei + BnBr
1000
1000
100
100
wt + B nBr mfd + B nBr
10
% Survival
Time Points (T30, T60, T90) 1. Centrifuge T0 eppies for 3 min. at 8000 rpm 2. Pipet out supernatant 3. Resuspend pellet in 500µL 0.85% saline. Store on ice. 4. Repeat for all time points
&''
MALDI/TOF/TOF-MS characterization of benzyl products with precursor ions m/z 226 (left, dA) and m/z 242 (right, dG) from calf thymus DNA modified with benzyl bromide. Courtesy Poguang Wang and Roger Giese.
Results
wt + BnBr nei + BnBr 10
Conclusions Genes showing sensitive to BnBr △dam, △mfd, △mutH
1 0
30
60
90
1 0
Time (min.)
30
60
Time (min.)
nth + BnBr
90
Genes not sensitive to BnBr △mutY, △nei, △nth
1000
% S urvival
Preparation of Overnights 1. 5mL LB + 1 colony strain from streaked plate 2. Duplicates made for each strain
Preparation of subcultures and dosing chemical 1. 7mL LB + overnight 2. Spin for 90 min at 37ºC 3. Use serial dilution to prepare 0.20mg/mL Benzyl Bromide
Prepare subcultures for pellet formation 1. Centrifuge subcultures at 3800rpm for 10 min. 2. Pour supernatant out and resuspend pellet in 1mL LB 3. 900µL à S eppie. 4. 90µL à T0 eppie
A-‐T base pair
% S urvival
Approach
Measure Subculture Growth 1. OD600= 0.1-‐0.2 2. Normalized subculture !"# volume=
%$Benzyl Bromide is extremely toxic by inhalation and by skin absorption • Corrosive to metals and tissue • Causes skin irritation, serious eye irritation, may cause respiratory irritation, and in large doses, can cause CNS depression • In fire, it forms vapor that is a powerful tear gas Exposure to BnBr may occur environmentally from: • Inhaling air contaminated by exhausts from cars using leaded gas • From its use in research
% S urvival
DNA alkylation is a process in which alkyl groups are covalently attached to DNA bases. The alkylated bases can arise from endogenous (via metabolism) or exogenous (via environmental exposure) sources. These DNA lesions can lead to mutations in DNA that can cause cancer. Alkylating agents are also used in cancer chemotherapy as cytotoxic agents. Understanding the DNA alkylation process and being able to identify the major DNA adducts that form from specific alkylating agents is crucial. The focus of our research is to understand and determine the survival of certain E. coli strains when treated with the alkylating agent, benzyl bromide, which is used industrially in foaming agents. We are carrying out assays using wildtype E. coli and single knockouts of forty-‐six specific genes associated with the numerous DNA repair processes to determine cellular survival when exposed to varied doses of benzyl bromide. We have identified some of the adducts formed in DNA from exposure to benzyl bromide. These assays help us identify how the bacterial cellular responses to benzyl bromide correlate with the identity and frequency of the adducts and also help us determine which genes are crucial for resistance to alkylation damage and to what degree.
My research works to find bacterial mutations that can bypass DNA damage caused by alkylating agents such as Benzyl Bromide
100
wt + B nBr nth + B nBr
10
1 0
30
60
Time (min.)
90
Acknowledgements
Characterization of Cellular Responses to DNA Alkylation in E. coli Jadesola Akanji, Macee Qi, Ariel Aiken, Alexis Hester, Caitlin Kramer, Becky Leifer, Mark Muenter, Penny Beuning
Opportunity
Impact
Prepare 96 Well Plate 1. 180µL 0..85% saline à rows C-‐G 2. 100µL of each time point à row B 3. Serial dilutions of 20µL from row BàC, CàD, DàE…
Chemical Dose Shock 1. 90µL serially dilute BnBr à S eppie 2. S eppies placed on shaker in 37ºC until time points
Plate Subcultures 1. On 50mL LB plate: 5µL of each row from G à B starting from bottom to top 2. Incubate inverted plates in 37ºC for 14-‐16 hours
G-‐C base pair
dam + BnBr
mutH + BnBr
1000
1000
wt + B nBr dam + B nBr
10
1 0
30
60
100
wt + BnBr mutH + BnBr
10
1
90
0
Time (min.)
30
60
% S urvival
% S urvival
100
1000
100
wt + BnBr mutY + BnBr 10
1
90
0
Time (min.)
mfd + BnBr
Data Analysis 1. Count colonies for each time point spot on LB plates 2. Record in lab notebook 3. Graph exposure time vs. % survival
mutY + BnBr
30
60
90
Time (min.)
nei + BnBr
1000
1000
100
100
wt + B nBr mfd + B nBr
10
% Survival
Time Points (T30, T60, T90) 1. Centrifuge T0 eppies for 3 min. at 8000 rpm 2. Pipet out supernatant 3. Resuspend pellet in 500µL 0.85% saline. Store on ice. 4. Repeat for all time points
&''
MALDI/TOF/TOF-MS characterization of benzyl products with precursor ions m/z 226 (left, dA) and m/z 242 (right, dG) from calf thymus DNA modified with benzyl bromide. Courtesy Poguang Wang and Roger Giese.
Results
wt + BnBr nei + BnBr 10
Conclusions Genes showing sensitive to BnBr △dam, △mfd, △mutH
1 0
30
60
90
1 0
Time (min.)
30
60
Time (min.)
nth + BnBr
90
Genes not sensitive to BnBr △mutY, △nei, △nth
1000
% S urvival
Preparation of Overnights 1. 5mL LB + 1 colony strain from streaked plate 2. Duplicates made for each strain
Preparation of subcultures and dosing chemical 1. 7mL LB + overnight 2. Spin for 90 min at 37ºC 3. Use serial dilution to prepare 0.20mg/mL Benzyl Bromide
Prepare subcultures for pellet formation 1. Centrifuge subcultures at 3800rpm for 10 min. 2. Pour supernatant out and resuspend pellet in 1mL LB 3. 900µL à S eppie. 4. 90µL à T0 eppie
A-‐T base pair
% S urvival
Approach
Measure Subculture Growth 1. OD600= 0.1-‐0.2 2. Normalized subculture !"# volume=
%$Benzyl Bromide is extremely toxic by inhalation and by skin absorption • Corrosive to metals and tissue • Causes skin irritation, serious eye irritation, may cause respiratory irritation, and in large doses, can cause CNS depression • In fire, it forms vapor that is a powerful tear gas Exposure to BnBr may occur environmentally from: • Inhaling air contaminated by exhausts from cars using leaded gas • From its use in research
% S urvival
DNA alkylation is a process in which alkyl groups are covalently attached to DNA bases. The alkylated bases can arise from endogenous (via metabolism) or exogenous (via environmental exposure) sources. These DNA lesions can lead to mutations in DNA that can cause cancer. Alkylating agents are also used in cancer chemotherapy as cytotoxic agents. Understanding the DNA alkylation process and being able to identify the major DNA adducts that form from specific alkylating agents is crucial. The focus of our research is to understand and determine the survival of certain E. coli strains when treated with the alkylating agent, benzyl bromide, which is used industrially in foaming agents. We are carrying out assays using wildtype E. coli and single knockouts of forty-‐six specific genes associated with the numerous DNA repair processes to determine cellular survival when exposed to varied doses of benzyl bromide. We have identified some of the adducts formed in DNA from exposure to benzyl bromide. These assays help us identify how the bacterial cellular responses to benzyl bromide correlate with the identity and frequency of the adducts and also help us determine which genes are crucial for resistance to alkylation damage and to what degree.
My research works to find bacterial mutations that can bypass DNA damage caused by alkylating agents such as Benzyl Bromide
100
wt + B nBr nth + B nBr
10
1 0
30
60
Time (min.)
90
Acknowledgements
