Difference between revisions of "Team:Warwick/Project2"

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<h4>Brixells</h4>
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<h4>Experiments</h4>
 
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Our team's aim is to create a tool box allowing the selective allocation of specific cell types to an engineered DNA scaffold, using zinc finger binding proteins on an E. coli model. Our research would allow for the self assembly of complex multi-type cell structures. The project will advance in progressive bands of complexity: designing and cloning the zinc finger coated E. coli cells, constructing DNA structure to allow for the cells to bind, further development of the zinc finger binding proteins allowing for multiple cell types to coexist on the DNA structure, and finally designing complex 3-D structures that the cells will be able to self assemble into. This has possible applications throughout medicine. Our research would contribute potentially to 3-D printing organic tissues, allowing for customised living tissues to be engineered.
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In order to attempt to achieve the goals that we set ourselves we came up with three experiments to test our theories.
 
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<H3>Experiment 1: Establishing Anchor Protein</H3>
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<dd class="active"><a href="#simple1">E.Coli</a></dd>
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<br>1. Golden gate assembly of plasmid backbones http://synbio.tsl.ac.uk/golden-gate-assembly-protocol/
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and 4 G-blocks (one of each candidate anchor protein and Zif268)
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<br>Waiting on enzymes (hopefully Friday/ Monday)
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<br>Lpp-OMPa is done
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We have chosen to use <i>E. coli</i> as the model for our concept for multiple reasons:
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<br>BclA, Pgsa, (INP) - waiting on enzymes to cut out anchor proteins
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<blockquote> It is the one of the most studied microorganisms, any changed we make to the genome are unlikely to have unexpected results. </blockquote>
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<blockquote> When made electrocompetent or chemically competetent it readily accepts plasmids. </blockquote>
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<br>2. Transform electrocompetent E. Coli cells with plasmid
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<blockquote> It has a rapid generation time so we can grow a lot of it in a short time period. </blockquote>
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<br>NB - IPTG needs to be added
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And most importantly:
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<br>Grow cells
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<blockquote> It can be cultured easily and inexpensively in a lab, so any mistakes we make when experimenting won't bankrupt us! </blockquote>
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<br>Prepared for microscopy (mounting medium)
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<br>DNA on glass (waiting epoxy, aminated oligo, fluorescent marker) - Friday/Monday
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<br>3. Bind antibodies to flag tag - measure using fluorescent detection mechanism
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<img src="" alt=""><b>Fun diagram of <i>E. coli!</i></b>
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<br>Antonia has components
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<br>4. Compare intensities - select anchor protein that fluoresces brightest
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<img src="https://static.igem.org/mediawiki/2015/e/e7/WarwickCell-ecoli.gif" alt=""><b>E.Coli Diagram</b>
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<br>Back-up:
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<br>Lyse cells to see if expression is taking place
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<br> <H3>Experiment 2: establishing zinc finger DNA binding proteins</H3>
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<br>1. Golden gate assembly of plasmid backbones and many G-blocks (one of each candidate zinc finger protein and the winning anchor protein) and with fluorescent protein code
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<br>2. Transform E. Coli with plasmid
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<br>3. Make wells of cognate DNA of the zinc fingers, introduce transformed E. Coli to the plate, leave to bind, wash plate and compare fluorescence of the E. Coli cells to determine which zinc finger binds strongest
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<br>4. Select 4 zinc fingers which bind strongest to their cognate DNA
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<br>Ben models = requires time points, do at the same time
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<br>Microscopy - do with both alive and dead
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<br>Test both coverslip or on slide
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<H3>Experiment 3: Checking colour</H3>
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<br>1. Transform E. Coli with each colour fluorescent protein and the anchor protein and each zinc finger protein
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- YFP, GFP, RFP - need to select a blue fluorescent protein
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<br>2. Test whether simple images can be made - green circle on a red square
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<br>3. Test colour mixing - mixing fluorescent cells to make colours not already present
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<br>4. Test complex images
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Revision as of 11:54, 25 August 2015

Warwick iGEM 2015

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