Difference between revisions of "Part:BBa K316001:Experience"
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<I>Imperial College iGEM 2010</I> | <I>Imperial College iGEM 2010</I> | ||
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| − | Characterization in r.p.u. of Pveg promoter | + | ===Characterization in r.p.u. of Pveg promoter=== |
Our experiment was performed on 96-well plates comparing four different populations of Catechol(2,3)dioxygenase expessing E.coli cells. These four different populations (initial O.D. 0.5) were: 1) XylE under the influence of J23101 promoter in pSB1C3 vector, 2) ) XylE under the influence of J23101 promoter in 3K3 vector, 3) XylE under the influence of pVeg promoter in pSB1C3 vector and 4) ) XylE under the influence of pVeg promoter in 3K3 vector. All 4 populations were grown under same condition and tested for the expression of catechol dioxygenase enzyme by addition 0.25mM catechol substrate. Reaction was monitored using spectrophotomer and the production of colored product was recorded. Production of HMS colored product is directly proportional to total enzyme presence. Since total enzyme presence depends on the strength of the promoter, this provides a means for quantification of the strength of promoters in relative promoter units. | Our experiment was performed on 96-well plates comparing four different populations of Catechol(2,3)dioxygenase expessing E.coli cells. These four different populations (initial O.D. 0.5) were: 1) XylE under the influence of J23101 promoter in pSB1C3 vector, 2) ) XylE under the influence of J23101 promoter in 3K3 vector, 3) XylE under the influence of pVeg promoter in pSB1C3 vector and 4) ) XylE under the influence of pVeg promoter in 3K3 vector. All 4 populations were grown under same condition and tested for the expression of catechol dioxygenase enzyme by addition 0.25mM catechol substrate. Reaction was monitored using spectrophotomer and the production of colored product was recorded. Production of HMS colored product is directly proportional to total enzyme presence. Since total enzyme presence depends on the strength of the promoter, this provides a means for quantification of the strength of promoters in relative promoter units. | ||
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Imperial College iGEM 2010 |
Characterization in r.p.u. of Pveg promoterOur experiment was performed on 96-well plates comparing four different populations of Catechol(2,3)dioxygenase expessing E.coli cells. These four different populations (initial O.D. 0.5) were: 1) XylE under the influence of J23101 promoter in pSB1C3 vector, 2) ) XylE under the influence of J23101 promoter in 3K3 vector, 3) XylE under the influence of pVeg promoter in pSB1C3 vector and 4) ) XylE under the influence of pVeg promoter in 3K3 vector. All 4 populations were grown under same condition and tested for the expression of catechol dioxygenase enzyme by addition 0.25mM catechol substrate. Reaction was monitored using spectrophotomer and the production of colored product was recorded. Production of HMS colored product is directly proportional to total enzyme presence. Since total enzyme presence depends on the strength of the promoter, this provides a means for quantification of the strength of promoters in relative promoter units. The results showed that pVeg promoter in pSB1C3 vector, a high copy plasmid, has an 1.62 r.p.u value and in 3K3 vector, a low copy plasmid an 0.79 r.p.u. value. These values were derived by dividing signal from the production of HMS by the pVeg promoter population of cells by signal from the standard promoter J23101 (r.p.u value of 1).Figure below:
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