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− | + | Week 1 (Aug 8): | |
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<br> | <br> | ||
− | + | As a part of our biotechnology pathway’s curriculum, we reviewed basic safety procedures and aseptic technique with our advisor Janet Standeven. | |
<br><br> | <br><br> | ||
</div> | </div> | ||
− | + | Week 2 (Aug 15): | |
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<br> | <br> | ||
− | + | 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 | + | |
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<br><br> | <br><br> | ||
</div> | </div> | ||
− | + | Week 3 (Aug 22): | |
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<br> | <br> | ||
− | + | 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. | |
<br><br> | <br><br> | ||
</div> | </div> | ||
− | + | Week 4 (Aug 29): | |
− | + | ||
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− | + | ||
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<br> | <br> | ||
− | + | 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. | |
<br><br> | <br><br> | ||
</div> | </div> | ||
− | + | Week 5 (Sep 5): | |
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<br> | <br> | ||
− | + | 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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<br><br> | <br><br> | ||
</div> | </div> | ||
− | + | >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. | ||
<br><br> | <br><br> | ||
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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 | + | Fall Break |
<br><br> | <br><br> | ||
</div> | </div> | ||
− | + | 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 | ||
<br><br> | <br><br> | ||
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− | + | Week 10 (Oct 10): | |
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<br> | <br> | ||
Ligation of P-lambda-R-LacI and B0034 | Ligation of P-lambda-R-LacI and B0034 | ||
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− | + | Week 11 (Oct 17): | |
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<br> | <br> | ||
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
![](https://static.igem.org/mediawiki/2016/2/26/T--LambertGA--purpleline.jpg)
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.
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.
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.
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.
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.
B0034 was hydrated from iGEM kit of parts
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