Presentation Title

Analyzing Manganese and Iron Levels in Knockout CSH104 Bacterial Strain

Presenter Information

Mariam MinasyanFollow
Paul LeeFollow

Faculty Mentor

Paul Lee

Start Date

23-11-2019 8:00 AM

End Date

23-11-2019 8:45 AM

Location

217

Session

poster 1

Type of Presentation

Poster

Subject Area

physical_mathematical_sciences

Abstract

Organisms have developed many different methods to defend themselves against oxidative damage. Oxidative damage, in the form of reactive oxygen species such as hydrogen peroxide (H2O2), superoxide (O2-) and the hydroxyl radical (OH∙), can disrupt a variety of molecules in living organisms, including proteins and DNA. We are interested in the latter, as oxidative damage to DNA can lead to detrimental mutations. One of those methods against oxidative damage, the import of manganese in response to peroxide stress, is poorly understood. In E. coli, when hydrogen peroxide levels increase enough to induce the OxyR operon, MntH, an NRAMP family protein, imports in manganese. This gene was knocked out in a reporter strain of E. coli, CSH104, using the Lambda-Red recombination method. The levels of Mn2+ in the knock out strain were compared to those in the parent strain using atomic absorption spectroscopy (AAS). As a control, iron levels were also monitored to ensure that undesirable mutations were not introduced. It was found that a freeze-thaw method in preparation followed by a nitric acid digestion was the ideal method for lysing the cells. When peroxide was introduced to the strain, the Mn2+ levels in the strain increased, in agreement with the literature. The data showed that the knockouts were successful, since the knockout contained lower Mn2+ levels than the parent strain. Iron levels were the same for the knockout and parent strain. Interestingly, manganese induction was observed even in the absence of peroxide induction suggesting that oxidative stress was being induced during normal experimental conditions.

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Nov 23rd, 8:00 AM Nov 23rd, 8:45 AM

Analyzing Manganese and Iron Levels in Knockout CSH104 Bacterial Strain

217

Organisms have developed many different methods to defend themselves against oxidative damage. Oxidative damage, in the form of reactive oxygen species such as hydrogen peroxide (H2O2), superoxide (O2-) and the hydroxyl radical (OH∙), can disrupt a variety of molecules in living organisms, including proteins and DNA. We are interested in the latter, as oxidative damage to DNA can lead to detrimental mutations. One of those methods against oxidative damage, the import of manganese in response to peroxide stress, is poorly understood. In E. coli, when hydrogen peroxide levels increase enough to induce the OxyR operon, MntH, an NRAMP family protein, imports in manganese. This gene was knocked out in a reporter strain of E. coli, CSH104, using the Lambda-Red recombination method. The levels of Mn2+ in the knock out strain were compared to those in the parent strain using atomic absorption spectroscopy (AAS). As a control, iron levels were also monitored to ensure that undesirable mutations were not introduced. It was found that a freeze-thaw method in preparation followed by a nitric acid digestion was the ideal method for lysing the cells. When peroxide was introduced to the strain, the Mn2+ levels in the strain increased, in agreement with the literature. The data showed that the knockouts were successful, since the knockout contained lower Mn2+ levels than the parent strain. Iron levels were the same for the knockout and parent strain. Interestingly, manganese induction was observed even in the absence of peroxide induction suggesting that oxidative stress was being induced during normal experimental conditions.