Supercoiling

From 2006.igem.org

Latest revision as of 16:10, 30 May 2006

How many flips would the normal negative supercoiling of a plasmid in E. coli allow? This depends on the superhelical density of the plasmid. We may be able to control this. It appears that two negative superhelical turns are used up in each recombination. Plasmids in the size range of 4000 bp usually contain between 8 and 12 negative supercoils.

What happens to supercoiling if we make the plasmid larger? If E coli controls the levels of superhelical density of plamids, then we may be able to control the number of flips using the size of the plasmid. If a plasmid of 4000 bp has 8 negative supercoils, then it may allow four recombinations. Addition of 2000 bp in the form of stuffer DNA (which doesn’t do anything) would increase the number of negative supercoils to 12, allowing six recombinations.

Expression of Fis is strongly dependent on superhelical density of DNA, highest with negative supercoiling, controls supercoiling homeostasis (Schneider et al. 2000).

What happens to supercoiling during the stationary phase, relax?

The expression of the Escherichia coli fis gene is strongly dependent on the superhelical density of DNA [http://www.blackwell-synergy.com/links/doi/10.1046/j.1365-2958.2000.02129.x/abs/]

E coli maintains constant superhelical energy in the DNA except during certain growth transitions, when changes in metabolism and gene expression are accompanied by changes in DNA supercoiling.

During the upshift of temperature from 30 to 42, 45, 47, or 50 degrees C, an increase in the level of supercoiling of a reporter plasmid was observed. The increase was inhibited in cells treated with chloramphenicol and novobiocin.

The level of supercoiling depends DNA topoisomerase I (TopI) and TopII (gyrase) and TopIV, and environmental variations alter this level.