Mutation Rates in E. coli MC4100 and Hfq -

Mutation Rates in E. coli MC4100 and Hfq-

Biomath Research - Summer ‘08

Glen Hamman

Jan Varada

Goals

To determine the effect of the Hfq protein in the deletion of quadruplex-forming DNA sequences.

If the Hfq protein is involved in removing the quadruplex structures, hfq+ cells with the protein should revert slower than hfq- cells without the protein.

Approach

Insert a DNA quadruplex-forming structure in the TET gene of the pBR325 plasmid.

Do a transformation into the E. coli HB101 strain, and plate to select AMP resistant colonies which have the plasmid.

Patch in grid in AMP and TET and select those that do not grow in TET because they have the quadruplex DNA structure.

Select those colonies, purify DNA and do transformations into the MC4100 and Hfq- strains and compare reversion rates.

DNA quadruplex structure

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pBR325 plasmid vector

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Results so far…

AMP plate TET plate

Results so far…

AMP plate CAP plate

Results so far…

Results so far…

To do…

Purify DNA from viable colonies and do transformations into the MC4100 and Hfq- strains.

Calculate the reversion frequency and complete the mutation rate experiment.

Follow up on the mathematical models describing the mutation behavior and compare predictions with experimental data.