Team:Groningen/Project/Construct

From 2013.igem.org

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<h1>Construct</h1>
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<h1>Constructs</h1>
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<br>
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<h3>Page lay-out</h3>
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<h3>Show our construct and give a short introduction</h3>
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<h3>Spider silk gene</h3>
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<br>What kind of spider silk gene is it
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<br>What are general problems with this gene
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<br>Where did we get it from.
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<br>Codon optimisation, solved the problems.
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<h3>Secretion</h3>
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<br>Why is it so important.
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<br> The use of existing pathway so not lots of trouble
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<br>The signal sequences are nice addition to the registry
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<br> how the pathway works and how the silk will be secreted
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<h3>Strep tag</h3>
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<br>Why this is needed
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<br>For what purposes it comes in handy
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<h2><i>amyE</i> intergrational backbone</h2>
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<h2>An <i>amyE</i> intergrational backbone</h2>
<p>
<p>
To transform <i>B. subtilis</i> with our developed biobricks we have improved the <i>amyE</i> intergrational backbone from Munich's iGEM team 2012 (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>) 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 (<a href="http://parts.igem.org/Part:BBa_K1085014">BBa_K1085014</a>)and can be induced with IPTG.<p>
To transform <i>B. subtilis</i> with our developed biobricks we have improved the <i>amyE</i> intergrational backbone from Munich's iGEM team 2012 (<a href="http://parts.igem.org/Part:BBa_K823023">BBa_K823023</a>) 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 (<a href="http://parts.igem.org/Part:BBa_K1085014">BBa_K1085014</a>)and can be induced with IPTG.<p>
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In figure 1 a close-up of the promoter is shown. Two lacO operaters 
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In figure 1 a close-up of the promoter is shown.  
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<p>The HyperSpank promoter has a single nucleotide change (G->T) at the -1 position. This increases the expersion levels but also causes leaky expersseion when IPTG is absence[1].
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To improve repression, a second lacO operator site has been inserted 71 bp upstream of the first. (David Rudner, Harvard Medical School)
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<h2>Silk concstructs</h2>
<h2>Silk concstructs</h2>
 +
<br>What kind of spider silk gene is it
 +
<br>What are general problems with this gene
 +
<br>Where did we get it from.
 +
<br>Codon optimisation, solved the problems.
<br>
<br>
<h2>Strep-tag</h2>
<h2>Strep-tag</h2>
 +
<br>Why this is needed
 +
<br>For what purposes it comes in handy
<br>
<br>
<h2>Signal peptide</h2>
<h2>Signal peptide</h2>
 +
<br>Why is it so important.
 +
<br> The use of existing pathway so not lots of trouble
 +
<br>The signal sequences are nice addition to the registry
 +
<br> how the pathway works and how the silk will be secreted
<br>
<br>

Revision as of 11:51, 1 October 2013

Constructs

An amyE intergrational backbone

To transform B. subtilis with our developed biobricks we have improved the amyE intergrational 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 expersion levels but also causes leaky expersseion when IPTG is absence[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: Hy_Spank promoter

Figure 2:

Promoter activity


Figure 3: The intensity in of ~40 cells in the GFP-channel, were analyzed from two pictures.
The average intensity (AU) from the cells are plotted above with the standard deviation.

GFPmg and GFP (BBa_E0840)


Figure 4:


Silk concstructs


What kind of spider silk gene is it
What are general problems with this gene
Where did we get it from.
Codon optimisation, solved the problems.

Strep-tag


Why this is needed
For what purposes it comes in handy

Signal peptide


Why is it so important.
The use of existing pathway so not lots of trouble
The signal sequences are nice addition to the registry
how the pathway works and how the silk will be secreted

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