Difference between revisions of "Team:LambertGA/Notebook"

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<h3>Notebook</h3>
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<p>
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Week 1 (Aug 8):
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<br>
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Safety procedures and aseptic technique was reviewed by our iGEM supervisor: Janet Standeven.
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<br><br>
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Week 2 (Aug 15):
 +
<br>
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This week was subjected to ligating two of our three parts of the genetic construct together: p-lambda-r/Lac I/ tspurple/ deg. Tag (LAA or DAS tags) + ROO1/ ClpXP/ CI
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<br>
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Note: no construct was ligated without a present degradation tag
 +
<br>
 +
After ligation was complete the the genetic constructs were then transformed into separate DH10 <i>E. coli</i> cells
 +
<br><br>
 +
Week 3 (Aug 22):
 +
<br>
 +
The previous transformation and ligation was unsuccessful due to the lack of ribosomal binding sites in the genetic constructs
 +
<br>
 +
We were required to re-digest, re-transform and re-ligate the p-lambda-r/ LacI/ TsPurple/ deg.tag(LAA or DAS) + ROO11/ ClpXP/ CI
 +
<br>
 +
Transformation and ligation of the third part: genetic construct without a degradation tag present
 +
<br><br>
 +
Week 4 (Aug 29):
 +
<br>
 +
The continuation of the re-ligations and re-transformations during week 3 were ultimately unsuccessful due to RFP( red fluorescent protein)  contamination
 +
<br>
 +
Miniprep and nanodrop of the genetic construct without a present degradation tag in the backbone: 1C3.
 +
<br><br>
 +
Week 5 (Sep 5):
 +
<br>
 +
Further detection of previous errors
 +
<br>
 +
Liquid culture of genetic construct without present degradation tag
 +
<br>
 +
Inoculation of RFP and previous TS purple colonies
 +
<br>
 +
Due to several unsuccessful procedures, we decide to religate p-lamba-r/LacI + ts purple (deg tag) and R0011/ClpXP + CI
 +
<br>
 +
The above ligations were also transformed
 +
<br><br>
 +
Week 6 (Sep 12)
 +
<br><br>
 +
Week 7 (Sep 19)
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<br><br>
 +
Week 8 (Sep 26)
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<br><br>
 +
Week 9 (Oct 3)
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<br><br>
 +
Week 10 (Oct 10)
 +
<br>
 +
Successful transformation of TsPurple and GFP constructs with variation of degradations tag (LAA, DAS and no degradation tag) and IPTG levels ( 1 uM, 10 uM, 100 Um, 1 mM)
 +
<br>
 +
Ligation
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<br><br>
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</p>
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</div></div></div></div>
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</section>
  
 
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Revision as of 13:39, 13 October 2016

Notebook


Week 1 (Aug 8):
Safety procedures and aseptic technique was reviewed by our iGEM supervisor: Janet Standeven.

Week 2 (Aug 15):
This week was subjected to ligating two of our three parts of the genetic construct together: p-lambda-r/Lac I/ tspurple/ deg. Tag (LAA or DAS tags) + ROO1/ ClpXP/ CI
Note: no construct was ligated without a present degradation tag
After ligation was complete the the genetic constructs were then transformed into separate DH10 E. coli cells

Week 3 (Aug 22):
The previous transformation and ligation was unsuccessful due to the lack of ribosomal binding sites in the genetic constructs
We were required to re-digest, re-transform and re-ligate the p-lambda-r/ LacI/ TsPurple/ deg.tag(LAA or DAS) + ROO11/ ClpXP/ CI
Transformation and ligation of the third part: genetic construct without a degradation tag present

Week 4 (Aug 29):
The continuation of the re-ligations and re-transformations during week 3 were ultimately unsuccessful due to RFP( red fluorescent protein) contamination
Miniprep and nanodrop of the genetic construct without a present degradation tag in the backbone: 1C3.

Week 5 (Sep 5):
Further detection of previous errors
Liquid culture of genetic construct without present degradation tag
Inoculation of RFP and previous TS purple colonies
Due to several unsuccessful procedures, we decide to religate p-lamba-r/LacI + ts purple (deg tag) and R0011/ClpXP + CI
The above ligations were also transformed

Week 6 (Sep 12)

Week 7 (Sep 19)

Week 8 (Sep 26)

Week 9 (Oct 3)

Week 10 (Oct 10)
Successful transformation of TsPurple and GFP constructs with variation of degradations tag (LAA, DAS and no degradation tag) and IPTG levels ( 1 uM, 10 uM, 100 Um, 1 mM)
Ligation