Under excessive amounts of hydrogen peroxide, a cell will experience oxidative stress, which defined as an imbalance between reactive oxygen species and antioxidant defenses. When it experiences this stress, the cell will activate genes to protect itself against oxidation.Escherichia coli, or E.coli reacts in part by activating genes that code for mntH, a manganese import protein. Manganese is a redox cofactor that is thought to help protect proteins from oxidative damage. The purpose of this research is to understand in depth the role manganese may have in DNA damage and repair. In this study, we have attempted to knock out the mntH gene in a reporter strain of E.coli, which is sensitive to GC to TA transversion DNA mutation. In order to investigate for the knockout of mntH, we utilized the technique of CRISPR/Cas 9. This method uses an endonuclease to modify a gene or other region DNA of interest. However, in this specific project, CRISPR/Cas 9 will be used to completely remove the mntH gene in our strain of interest, CSH104. To perform CRISPR on our strains, a 20 base pair crRNA sequence will be cloned into the crRNA expression plasmid using either traditional cloning techniques or inverse PCR. This plasmid will be used to transform electrocompetent cells. The secondary plasmid containing the Cas9 expression system will then be used to transform these cells, resulting in a strain that contains the complete CRISPR/Cas9 system. To verify the loss of manganese import function, a measurement of the manganese content of both wild-type and knockout will then be evaluated through the technique of Atomic Absorption (AA).

Construction of mnth knockout in Escherichia coli strain CSH104 using CRISPR/Cas9 and verification of manganese concentration using atomic apsorption spectroscopy

2019 Spring National Meeting

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