Team:EPF-Lausanne/Our Project/T7 promoter variants

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With T7 and T7-lac promoter variants in hand, we want to characterize their relative strengths. To obtain the right set-up, we transform the promoter variant plasmids into a different strain of E. coli called BL21. These cells have a mutation in the promoter for the lacI gene. As a result of this mutation, LacI protein is overproduced and is found abundantly in these cells.  
With T7 and T7-lac promoter variants in hand, we want to characterize their relative strengths. To obtain the right set-up, we transform the promoter variant plasmids into a different strain of E. coli called BL21. These cells have a mutation in the promoter for the lacI gene. As a result of this mutation, LacI protein is overproduced and is found abundantly in these cells.  
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[[File:bl21_basic.png]]
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[[File:basic_bl21.png]]
In this same strain, the gene for the T7 RNA polymerase is preceded by a lac operator. Since LacI is a repressor and is strongly present in BL21 cells, the production of T7 RNA polymerases is severely repressed. With very few T7 RNA polymerases available, there is very little recognition and binding of T7 promoters and consequently very little expression of the gene driven by the T7 promoter. Moreover, the expression of a gene driven by a T7-lac promoter would be even less, since the presence of LacI would block any action of the T7 RNA polymerase.  
In this same strain, the gene for the T7 RNA polymerase is preceded by a lac operator. Since LacI is a repressor and is strongly present in BL21 cells, the production of T7 RNA polymerases is severely repressed. With very few T7 RNA polymerases available, there is very little recognition and binding of T7 promoters and consequently very little expression of the gene driven by the T7 promoter. Moreover, the expression of a gene driven by a T7-lac promoter would be even less, since the presence of LacI would block any action of the T7 RNA polymerase.  

Revision as of 10:29, 19 September 2011