Team:Groningen/Project/Construct

From 2013.igem.org

(Difference between revisions)
Line 1: Line 1:
<html>
<html>
-
<head>
 
-
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN"
 
-
"http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
 
-
 
-
<script type="text/javascript" src="http://ajax.googleapis.com/ajax/libs/jquery/1.3.2/jquery.min.js"></script>
 
-
 
-
<script type="text/javascript" src="scrolltopcontrol.js">
 
-
 
-
/***********************************************
 
-
* Scroll To Top Control script- © Dynamic Drive DHTML code library (www.dynamicdrive.com)
 
-
* This notice MUST stay intact for legal use
 
-
* Visit Project Page at http://www.dynamicdrive.com for full source code
 
-
***********************************************/
 
-
 
-
</script>
 
-
</head>
 
<h1>Production backbone</h1>
<h1>Production backbone</h1>

Revision as of 22:40, 4 October 2013

Production backbone

An amyE locus integration backbone

To transform B. subtilis with our developed silk biobricks we have improved the amyE integrational backbone from Munich's iGEM team 2012 (BBa_K823023) that had no inducible promoter. We've added the HyperSpank IPTG inducible promoter from Rudner's Lab 2004. This promoter is placed right in front of the prefix, so any biobrick can be inserted in this backbone (BBa_K1085014) and can be induced with IPTG.

In figure 1 a close-up of the promoter is shown.

The HyperSpank promoter has a single nucleotide change (G->T) at the -1 position. This increases the expression levels but also causes leaky expression when IPTG is absent [1]. To improve repression, a second lacO operator site has been inserted 71 bp upstream of the first (David Rudner, Harvard Medical School).

Figure 1: The HyperSpank promoter with in orange the lacO operators, in red the -35, -10, +1 signals and in green the prefix.

Figure 2: BBa_K1085014 with: The amp gene for ampicillin resistance in E. coli (not shown); The cat gene for chloramphenicol resistance in B. subtilis; The HyperSpank promoter with its repressor lacI; The amyE up- and downstream fragments for intergration in the amyE locus.

For the validation of this backbone, click here.

References

[1]      Quisel, John D., William F. Burkholder, and Alan D. Grossman. "In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis." Journal of bacteriology 183.22 (2001): 6573-6578.

Retrieved from "http://2013.igem.org/Team:Groningen/Project/Construct"