Difference between revisions of "Part:BBa K316004:Experience"

 
m
 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
Line 5: Line 4:
  
 
===Applications of BBa_K316004===
 
===Applications of BBa_K316004===
 +
 +
K316004 is the XylE expressing version of the <bbpart>I20260</bbpart> reference promoter construct and can be used as a 1 strength reference object to characterise promoters according to the methods put forward by Kelly et al. (2009)<cite>1</cite>
 +
 +
  
 
===User Reviews===
 
===User Reviews===
 
<!-- DON'T DELETE --><partinfo>BBa_K316004 StartReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K316004 StartReviews</partinfo>
 +
{|width='80%' style='border:1px solid gray'
 +
|-
 +
|width='10%'|
 +
<partinfo>BBa_K316001 AddReview 5</partinfo>
 +
<I>Imperial College iGEM 2010</I>
 +
|width='60%' valign='top'|
 +
===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.
 +
 +
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).
 +
 +
 +
[[Image:Untitled 5.jpg.png|400px|Graph showing the ratio of pVeg/J23101 signals in pSB1C3 and 3K3 vectors]]
 +
|}
 
<!-- Template for a user review
 
<!-- Template for a user review
 
{|width='80%' style='border:1px solid gray'
 
{|width='80%' style='border:1px solid gray'
Line 19: Line 37:
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
 
<!-- DON'T DELETE --><partinfo>BBa_K316004 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K316004 EndReviews</partinfo>
 +
 +
<biblio>
 +
#1 pmid=19298678
 +
</biblio>

Latest revision as of 01:23, 28 October 2010

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K316004

K316004 is the XylE expressing version of the I20260 reference promoter construct and can be used as a 1 strength reference object to characterise promoters according to the methods put forward by Kelly et al. (2009)1


User Reviews

UNIQ518d4cdb521324c9-partinfo-00000001-QINU

•••••

Imperial College iGEM 2010

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.

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).


Graph showing the ratio of pVeg/J23101 signals in pSB1C3 and 3K3 vectors

UNIQ518d4cdb521324c9-partinfo-00000003-QINU

<biblio>

  1. 1 pmid=19298678

</biblio>