Difference between revisions of "Team:UNOTT/Experiments"

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<p> The highest and lowest performing promoters were chosen to give the most easily visible result. Promoter E and Promoter 4. Promoter 4 gives a high expression of fluorescent proteins, as shown by our promoter library findings. The promoters were then attached to each reporter protein CFP, RFP and GFP to form six "brick" variants. After amplification of the bricks produced, seven products combinations were ligated to a low copy backbone as controls, in addition to a set of "random ligations" These ligations have only one ligation slot (due to availability of restriction cut sites) per reporter type, leaving random chance to produce a combination of all the possible variants. e= empty promoter, h= high expression promoter</p>
 
<p> The highest and lowest performing promoters were chosen to give the most easily visible result. Promoter E and Promoter 4. Promoter 4 gives a high expression of fluorescent proteins, as shown by our promoter library findings. The promoters were then attached to each reporter protein CFP, RFP and GFP to form six "brick" variants. After amplification of the bricks produced, seven products combinations were ligated to a low copy backbone as controls, in addition to a set of "random ligations" These ligations have only one ligation slot (due to availability of restriction cut sites) per reporter type, leaving random chance to produce a combination of all the possible variants. e= empty promoter, h= high expression promoter</p>
 
<center><img src="https://static.igem.org/mediawiki/2017/a/a1/T--UNOTT--brickstitching.jpeg"></center>
 
<center><img src="https://static.igem.org/mediawiki/2017/a/a1/T--UNOTT--brickstitching.jpeg"></center>
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<p> These bricks were stuck </p>
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<p> These bricks were ligated with the backbone as follows: </p>
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<li> eRFP, eGFP, eCFP - Low expression in all reporters</li>
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<li> eRFP-eGFP-eCFP - Low expression in all reporters</li>
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<li> hRFP, hGFP, hCFP - High expression in all reporters</li>
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<li> hRFP-hGFP-hCFP - High expression in all reporters</li>
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<li> hRFP, eGFP, eCFP - Low expression in GFP and CFP, but High expression of RFP</li>
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<li> hRFP-eGFP-eCFP - Low expression in GFP and CFP, but High expression of RFP</li>
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<li> eRFP, hGFP, eCFP - Low expression in RFP and CFP, but High expression of GFP</li>
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<li> eRFP-hGFP-eCFP - Low expression in RFP and CFP, but High expression of GFP</li>
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<li> eRFP, eGFP, hCFP - Low expression in RFP and GFP, but High expression of CFP</li>
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<li> eRFP-eGFP-hCFP - Low expression in RFP and GFP, but High expression of CFP</li>
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<li> eRFP, eGFP, eCFP, hRFP, hGFP, hCFP - As both variants of each reporter brick available, every combination is possible. </li>
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<li> eRFP or hRFP-eGFP or hGFP-eCFP or hCFP - As both variants of each reporter brick available, every combination is possible. </li>
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<p> This produced a control for each reporter in isolation. It also produced a control for all reporters on either highest expression or lowest expression. This finally also produced a random set of colonies for isolation for comparison and test for true randomness.</p>
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<p> This produced a control for each reporter fluorescent protein in isolation. It also produced a control for all reporters on either highest expression or lowest expression. This finally also produced a random set of colonies for isolation (after transformation) for comparison and test for true randomness.</p>
 
<h1>Results</h1>
 
<h1>Results</h1>
  

Revision as of 18:23, 1 November 2017





EXPERIMENTS:

STEP 1: Create guideRNA Plasmid

STEP 2: Create Reporter Plasmid

STEP 3: Promoter Library

STEP 4: Random Ligations

STEP 5: Freeze Drying & Revival

STEP 6: CRISPRi & gRNA Efficiency