Difference between revisions of "Team:LambertGA/Notebook"

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<button class="accordion">Week 1 (Aug 8):</button>
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Week 1 (Aug 8):
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Safety procedures and aseptic technique was reviewed by our iGEM supervisor: Janet Standeven.
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As a part of our biotechnology pathway’s curriculum, we reviewed basic safety procedures and aseptic technique with our advisor Janet Standeven.
 
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<button class="accordion">Week 2 (Aug 15):</button>
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Week 2 (Aug 15):
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This week was subjected to ligating two of our three parts of the genetic construct together: <b>P--Lambda-R--LacI--Tspurple (LAA/DAS) and ROO11--ClpXP--CI</b>
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Our focus this week was to ligate two of the three parts in our genetic construct: P-lambda-R--LacI--TS Purple--LAA/DAS and R0011--ClpXP--CI; however, we did not ligate the part without any degradation tag.  After the ligations, the constructs were transformed into DH10 E. coli cells, but we were unsuccessful.  
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After ligation was complete the the genetic constructs were then transformed into separate DH10 <i>E. coli</i> cells  
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Results: Unsuccessful for unknown reasons. Looking back it was most likely a procedure-error or a lack of a B0034 RBS binding site
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<button class="accordion">Week 3 (Aug 22):</button>
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Week 3 (Aug 22):
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The previous transformation and ligation was unsuccessful due to the lack of ribosomal binding sites in the genetic constructs
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We were required to re-digest, re-transform and re-ligate the <b>P--Lambda-R--LacI--Tspurple (LAA/DAS) and ROO11--ClpXP--CI</b>
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Transformation and ligation of the third part: genetic construct without a degradation tag on the Tspurple Protein
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Due to the unsuccessful transformations seen the previous week, we re-attempted our workflow (digest, ligation, transformation) of both parts.  In addition, we ligated and transformed our third construct: P-lambda-R--LacI--TS Purple and R0011--ClpXP--CI.
 
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Week 4 (Aug 29):
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<br>
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The continuation of the re-ligations and re-transformations during week 3 were ultimately unsuccessful due to RFP(red fluorescent protein) contamination and/or lack of functioning genetic construct
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Minipreped and nanodroped the genetic construct without a present degradation tag in the backbone 1C3.
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Transformations were ultimately unsuccessful because of RFP (red fluorescent protein) contamination.  In addition, we miniprepped and nanodropped our genetic construct without the present degradation tag, which was in the 1C3 backbone.
 
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Week 5 (Sep 5):
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Further detection of previous errors
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Liquid culture of genetic construct without degradation tag
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Inoculation of RFP and previous TS purple colonies
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Performed digests of p-lambda-r/LacI,  Ts purple (no deg tag/ DAS/ LAA), R0011-ClpXP-CI, R0011-ClpXP, and CI.
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Show picture of gel
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Due to several failures over the past few weeks, we attempted to detect previous errors by re-ligating and re-transforming the parts together using a different stock of P-lambda-R--LacI that was previously confirmed over the summer.  Our results indicated faint, purple cells for our construct (P-lambda-R--LacI--TS Purple--R0011--ClpXP--CI), but no color was shown for the DAS and LAA cells after the transformation.  In addition, we inoculated liquid cultures of our genetic constructs from the previous week’s transformations, including the genetic construct without the present degradation tag, the RFP colonies, and previous TS Purple colonies.  Digests were performed of P-lambda-R--LacI--TS Purple--no degradation tag/DAS/LAA, R0011--ClpXP, and CI.  After analyzing the gel, we concluded the TS Purple with no degradation tag/DAS/LAA, R0011--ClpXP, and CI were of expected sizes, but the P-lambda-R--LacI and R0011--ClpXP--CI digests were unsuccessful. Lastly, the following backbones were successfully digested for future ligations: 1A3, 1C3, 1K3, and 1T3.
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Conclusion: TsPurple(no deg tag/ DAS/LAA), R0011-ClpXP, and CI digests were of expected sizes and p-lambda-r-LacI, and R0011-ClpXP-CI digests were unsuccessful
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Digested 1A3, 1C3, 1K3, and 1T3 backbones for future ligations
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Show gel
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Conclusion: all successful
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Due to several unsuccessful procedures, we re-ligated p-lamba-r/LacI + Ts purple (no deg tag/DAS/LAA) and R0011/ClpXP + CI using a different stock of p-lambda-r-LacI that was previously confirmed
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The above ligations were also transformed
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Results: very faint cells for p-lamba-r/LacI + Ts purple (no deg tag) and no color for DAS or LAA cells
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>Week 6 (Sep 12):
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We sent out constructs for sequencing and found that our constructs did not have an RBS between LacI and Tspurple, so our cells would turn purple via read-through transcription, meaning only cells with TsPurple (no deg Tag) would be slightly purple but a deg tagged TsPurple would result in no color at all.
 
We sent out constructs for sequencing and found that our constructs did not have an RBS between LacI and Tspurple, so our cells would turn purple via read-through transcription, meaning only cells with TsPurple (no deg Tag) would be slightly purple but a deg tagged TsPurple would result in no color at all.
 
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Week 7 (Sep 19):
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Digestion of TsPurple (no deg tag/DAS/LAA) and ligation into 1C3 vector. Then we transformed into NEB 10-beta cells
 
Digestion of TsPurple (no deg tag/DAS/LAA) and ligation into 1C3 vector. Then we transformed into NEB 10-beta cells
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Week 8 (Sep 26):
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Fall Break (We did stuff here)
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Fall Break  
 
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Week 9 (Oct 3):
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Decided to use premade GFP constructs as a proof-of-concept while simultaneously building our Tspurple constructs  
 
Decided to use premade GFP constructs as a proof-of-concept while simultaneously building our Tspurple constructs  
 
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Week 10 (Oct 10):
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Ligation of P-lambda-R-LacI and B0034
 
Ligation of P-lambda-R-LacI and B0034
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Successful miniprep of R0011--ClpXP--CI and P-lambda-R--LacI--GFP--ClpXP--CI
 
Successful miniprep of R0011--ClpXP--CI and P-lambda-R--LacI--GFP--ClpXP--CI

Revision as of 19:42, 19 October 2016



Notebook


Week 1 (Aug 8):
As a part of our biotechnology pathway’s curriculum, we reviewed basic safety procedures and aseptic technique with our advisor Janet Standeven.

Week 2 (Aug 15):
Our focus this week was to ligate two of the three parts in our genetic construct: P-lambda-R--LacI--TS Purple--LAA/DAS and R0011--ClpXP--CI; however, we did not ligate the part without any degradation tag. After the ligations, the constructs were transformed into DH10 E. coli cells, but we were unsuccessful.

Week 3 (Aug 22):
Due to the unsuccessful transformations seen the previous week, we re-attempted our workflow (digest, ligation, transformation) of both parts. In addition, we ligated and transformed our third construct: P-lambda-R--LacI--TS Purple and R0011--ClpXP--CI.

Week 4 (Aug 29):
Transformations were ultimately unsuccessful because of RFP (red fluorescent protein) contamination. In addition, we miniprepped and nanodropped our genetic construct without the present degradation tag, which was in the 1C3 backbone.

Week 5 (Sep 5):
Due to several failures over the past few weeks, we attempted to detect previous errors by re-ligating and re-transforming the parts together using a different stock of P-lambda-R--LacI that was previously confirmed over the summer. Our results indicated faint, purple cells for our construct (P-lambda-R--LacI--TS Purple--R0011--ClpXP--CI), but no color was shown for the DAS and LAA cells after the transformation. In addition, we inoculated liquid cultures of our genetic constructs from the previous week’s transformations, including the genetic construct without the present degradation tag, the RFP colonies, and previous TS Purple colonies. Digests were performed of P-lambda-R--LacI--TS Purple--no degradation tag/DAS/LAA, R0011--ClpXP, and CI. After analyzing the gel, we concluded the TS Purple with no degradation tag/DAS/LAA, R0011--ClpXP, and CI were of expected sizes, but the P-lambda-R--LacI and R0011--ClpXP--CI digests were unsuccessful. Lastly, the following backbones were successfully digested for future ligations: 1A3, 1C3, 1K3, and 1T3.

>Week 6 (Sep 12): We sent out constructs for sequencing and found that our constructs did not have an RBS between LacI and Tspurple, so our cells would turn purple via read-through transcription, meaning only cells with TsPurple (no deg Tag) would be slightly purple but a deg tagged TsPurple would result in no color at all.

B0034 was hydrated from iGEM kit of parts

Week 7 (Sep 19): Digestion of TsPurple (no deg tag/DAS/LAA) and ligation into 1C3 vector. Then we transformed into NEB 10-beta cells

Digestion of B0034 and TsPurple (no deg tag/DAS/LAA)
Results: all successful

Ligation of B0034 & TsPurple (no deg tag/DAS/LAA)

Transformation of B0034 and TsPurple (no tag/DAS/LAA)
Results: Successful growth

Week 8 (Sep 26): Fall Break