Team:Cambridge/Project/In Vivo

From 2011.igem.org

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=Objective One - Express reflectin in E. coli=
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=Expressing reflectin in E. coli=
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Our first objective was to try to express reflectin, in any shape or form, in E. coli. Ideally, we'd be able to express reflectin in the bacteria at a range of levels using a single construct, so we can both overexpress (for ''in vitro'' studies) or underexpress (to promote ''in vivo'' folding) straightforwardly. In order to do this, we had to create an expression plasmid for reflectin with which we could transform the bacterial cells. Hence, we had to isolate a reflectin gene sequence, choose a suitable promotor and join them on an appropriate backbone in order to engineer what we wanted. These steps are outlined below.
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To help pave the way for the manipulation of living structural colour, we investigated the properties of recombinant reflectins ''in vivo''. We designed multiple expression constructs to allow us to study reflectin under different conditions. An outline of our plan is given below.
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[[File:CAM_invivo_flow.png | centre | 670px]]
[[File:CAM_invivo_flow.png | centre | 670px]]
==Isolating the reflectin gene==
==Isolating the reflectin gene==
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This was more complicated than we initially expected. Our first idea was to attempt to clone reflectin genes from genomic DNA, extracted from the same squid specimens that we dissected for our [https://2011.igem.org/Team:Cambridge/Project/prelim preliminary observations]. We started with a very crude genomic extraction protocol, but unfortunately we did not manage to successfully clone reflectin using this method (details of the experiment can be found [https://2011.igem.org/Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample here]). We then attempted two cleaner genomic extraction protocols (described [https://2011.igem.org/Team:Cambridge/Experiments/Squid_Dissection_and_Tissue_Sample_Improved_Protocol here]), but unfortunately we remained unsuccessful. After some discussion with Dr. Wendy Goodson, a researcher who was one of the first to study reflectin, we found out that cloning from cephalopod genomic DNA is notoriously difficult, although the cause for this is not understood. She then very kindly offered to send us a quantity of cloning plasmids containing reflectin genes that she has worked with in the past; in this way we obtained genes for reflectin A1, A2 and 1B that had already been codon optimised for expression in E. coli. The amplification of these plasmids is described [https://2011.igem.org/Team:Cambridge/Experiments/Transformation_of_E.coli_with_Plasmids_from_Wendy here].
 
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==Selecting the promotor==
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We initially attempted to clone reflectin genes from ''Loligo pealeii'' genomic DNA, but this proved difficult - extracting squid DNA is notoriously difficult and we were unable to obtain samples fresh enough to create a cDNA library.  We are particularly grateful to Wendy Crookes-Goodson for her kind donation of plasmids containing reflectins codon-optimised for ''E. coli''.
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==Selecting the backbone==
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==Designing expression constructs==
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==Creating the expression plasmid==
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Promoter choice, copy number - justification
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The expression plasmids were created by Gibson Assembly.
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==Expression in E. coli==
 
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Hopefully coming soon!
 
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==Periplasmic Export==
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Revision as of 11:47, 16 September 2011

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Contents

Expressing reflectin in E. coli

To help pave the way for the manipulation of living structural colour, we investigated the properties of recombinant reflectins in vivo. We designed multiple expression constructs to allow us to study reflectin under different conditions. An outline of our plan is given below.

CAM invivo flow.png

Isolating the reflectin gene

We initially attempted to clone reflectin genes from Loligo pealeii genomic DNA, but this proved difficult - extracting squid DNA is notoriously difficult and we were unable to obtain samples fresh enough to create a cDNA library. We are particularly grateful to Wendy Crookes-Goodson for her kind donation of plasmids containing reflectins codon-optimised for E. coli.

Designing expression constructs

Promoter choice, copy number - justification


Periplasmic Export


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