Part:BBa_K891234:Experience

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Characterization

We expected Topoismerase D168A to nick at the YCCTT site and covalently bind. The below results demonstrates successful over-expression of this biobrick in E.coli cells and shows nicking activity.

Mass Spec Verification of Protein Expression

Peak 2428 demonstrates successful over-expression of Topoisomerase D168A, a protein toxic to E.coli cells.

Gel confirmation of Topoisomerase D168A interaction with target plasmid

Lane 5 (GFPT2+TopoD168A) demonstrates multi-nicked plasmids which run faster on the gel than Lane 2(GFPT2). The band also smears upwards, demonstrating loss of supercoiling in plasmid GFPT2.

Bradford Assay

This Bradford assay demonstrates over-expression of Topo D168A mutant over background signal.

Gel result of varying amount of Topoisomerase YCCTT sites

Omega fragment possesses no topoisomerase sites. It had the least amount of D168A interaction as shown on the gel. Topoisomerase coding sequence possesses four TCCTT sits on the antisense strand. The G1 PCR product is decorated with YCCTT sites on both the sense and antisense strands and experienced the greatest amount of nicking.

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