Difference between revisions of "Part:BBa K2203000"

(→‎Characterization of lysate expressing the gamS protein)
(→‎GamS use in cell-free systems)
 
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===GamS use in cell-free systems===
 
===GamS use in cell-free systems===
 
This part has a demonstrated positive effect on protein synthesis in a cell-free
 
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
+
chassis. GamS can be added to a cell-free reaction system in several ways: By adding it in its purified
 
synthesized protein form, or by adding simply its coding sequence to the cell-free reaction, or (in the
 
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
 
case of a self-made cell lysate) by making the lysate from cells which were transformed with the
−
GamS-plasmid.  
+
GamS-plasmid and thus subsequently have produced the GamS protein before lysis.  
  
−
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.
+
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 results in less protein 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.
+
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 : the same effect could 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 were created by transformating Top10 cells with a plasmid coding for the gamS protein.
 
For the first and last example, see experimental results below.
 
For the first and last example, see experimental results below.
  
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===Characterization of lysate expressing the gamS protein===
 
===Characterization of lysate expressing the gamS protein===
 
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Latest revision as of 02:05, 2 November 2017


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 purified 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 and thus subsequently have produced the GamS protein before lysis.

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 results in less protein 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 : the same effect could 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 were created by transformating Top10 cells with a plasmid coding for the gamS protein. For the first and last example, see experimental results below.


Characterization of lysate expressing the gamS protein

Expression of EGFP was measured on a plate reader. EGFP template was added to a usual lysate reaction as described here. Reactants were kept at 37°C for five hours. Compared are expression levels of lysate without either GamS protein or Top10-GamS lysate, lysate with GamS protein and lysate mixed with Top10-GamS lysate.

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Figure 1: Protein expression in M15-T7 lysate.
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Figure 2: Protein expression in M15-T7 lysate: end point measurements after 6 hours .

It can be concluded that addition of GamS in any form (protein or in Top10-GamS lysate) results in more fluorescence and thus higher reaction efficiency.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 298
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]