Difference between revisions of "Part:BBa K2203000"

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gamS protein

GamS is a protein that protects linear DNA templates from degradation in a cell- free environment. It is used to enhance cell-free protein synthesis by inhibiting exonuclease activity.

GamS use in cell-free systems

This part has a demonstrated positive effect on protein synthesis in a cell-free chassis. GamS can be added to a cell-free reaction system in several ways: By adding it in its synthesized protein form, or by adding simply its coding sequence to the cell-free reaction, or (in the case of a self-made cell lysate) by making the lysate from cells which were transformed with the GamS-plasmid. Linear DNA template that is added to a cell-free expression system is at high risk of being cut by an exonuclease still present in the lysate. This has as a result that less protein is being produced. When adding the DNA in plasmid form this issue can be prevented. The gamS protein of phage lambda is an inhibitor of the exonuclease. When added purified to a cell-free system containing linear DNA template, protein expression increases substantially. As purified gamS is quite expensive, another way was explored : this effect could also be achieved when the production of the gamS protein was induced in cells before the subsequent lysis of the cell culture. We showed this by mixing two lysates: M15-T7, which is optimised for protein production in cell-free, and top10-gamS, which contains gamS protein. Bacterial cells used for Top10-gamS lysate production was done by transformation of Top10 cells with a plasmid coding for the gene gamS. For the first and last example, see experimental results below.

Characterization of lysate expressing the gamS protein

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Sequence and Features