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Team:Groningen/Project/Construct
From 2013.igem.org
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 expression levels but also causes leaky expression 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)
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| Figure 1: Hy_Spank promoter |
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| Figure 2: |
Promoter activity
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| Figure 3: In (a) 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. In (b) a biobricked GFP (BBa_E0840) is shown with GFP in one plot. |
GFPmg and GFP (BBa_E0840)
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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
