Team:Caltech/week4

From 2014.igem.org

(Difference between revisions)
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<b>Export Systems</b>
<ul><li>PCR extracted vector backbone from pKS001 with Biobrick prefix & suffix overhangs</li>
<ul><li>PCR extracted vector backbone from pKS001 with Biobrick prefix & suffix overhangs</li>
     <li>PCR purification of PCR product and then 1 hour incubation at 37&deg;C with DPN1 enzyme to remove any remaining sticky ends</li>
     <li>PCR purification of PCR product and then 1 hour incubation at 37&deg;C with DPN1 enzyme to remove any remaining sticky ends</li>
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     <li>Transformation of assemblies into JM109. Plated on ampicillin plates and incubated at 37&deg;C overnight</li>
     <li>Transformation of assemblies into JM109. Plated on ampicillin plates and incubated at 37&deg;C overnight</li>
</ul>
</ul>
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<b>Combinatorial Promoters</b>
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<ul><li>Set up liquid cultures of the colonies that yielded promising results in the colony PCR conducted last Thursday; cultures incubated overnight</li></ul>
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<b>Export Systems</b>
<ul><li>Picked 4 colonies per plate incubated last night and resuspended the bacteria in 10 uL H<sub>2</sub>O (20 colonies picked total, 4 representing each of the 5 export domains)</li>
<ul><li>Picked 4 colonies per plate incubated last night and resuspended the bacteria in 10 uL H<sub>2</sub>O (20 colonies picked total, 4 representing each of the 5 export domains)</li>
     <li>Colony PCR was then run on 1 uL samples of the resuspensions</li>
     <li>Colony PCR was then run on 1 uL samples of the resuspensions</li>
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     <li>Picked 10 resuspensions (2 of each export domain) and used them to inoculate 5 mL liquid cultures, which were placed in the 37&deg;C shaker overnight</li>
     <li>Picked 10 resuspensions (2 of each export domain) and used them to inoculate 5 mL liquid cultures, which were placed in the 37&deg;C shaker overnight</li>
</ul>
</ul>
 +
<b>Combinatorial Promoters</b>
 +
<ul><li>Miniprepped liquid cultures grown up last night to extract plasmids</li>
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    <li>Sent in the resulting plasmids for sequencing</li></ul>
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<b>Export Systems</b>
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<ul><li>Discovered that the reverse primer used in colony PCR last night had no binding site on the plasmids we had assembled (pTG001 constructs)</li>
 +
    <li>Miniprepped liquid cultures grown up last night to extract plasmids
 +
    <ul><li>Not all bacteria were able to be resuspended after pelleting, so resulting DNA yields for 6 of the 10 liquid cultures grown were very low (10-25 ng/uL)</li></ul>
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    </li>
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</ul>
 +
<b>Combinatorial Promoters</b>
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<ul><li>Analyzed sequencing results and grew more liquid cultures of the colonies containing properly assembled plasmids</li>
</ul>
</ul>
<br>
<br>

Revision as of 18:10, 10 July 2014
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Notebook

Overview

Week 1

Week 2

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Week 12

Week Four

Monday, 7/7/14

 Export Systems
  • PCR extracted vector backbone from pKS001 with Biobrick prefix & suffix overhangs
  • PCR purification of PCR product and then 1 hour incubation at 37°C with DPN1 enzyme to remove any remaining sticky ends
  • PCR purification of DPN1-digested product
  • Ran a gel that confirmed presence of ~3000bp-long DNA fragment in both DPN1-digested and undigested PCR products
  • Gibson Assembly of the PCR fragments with the geneblocks ordered 2 weeks ago to form the 4 exportDomain-comX-mNeonGreen constructs (incorporating GeneIII, OmpA, PelB, and TorA as the export domains in the N-terminus) and the 1 comX-mNeonGreen-HlyA construct, all in the vector backbone
  • Transformation of assemblies into JM109. Plated on ampicillin plates and incubated at 37°C overnight
Combinatorial Promoters
  • Set up liquid cultures of the colonies that yielded promising results in the colony PCR conducted last Thursday; cultures incubated overnight

Tuesday, 7/8/14

 Export Systems
  • Picked 4 colonies per plate incubated last night and resuspended the bacteria in 10 uL H2O (20 colonies picked total, 4 representing each of the 5 export domains)
  • Colony PCR was then run on 1 uL samples of the resuspensions
    • Unfortunately, PCR products showed no DNA constructs on running through a gel
  • Picked 10 resuspensions (2 of each export domain) and used them to inoculate 5 mL liquid cultures, which were placed in the 37°C shaker overnight
Combinatorial Promoters
  • Miniprepped liquid cultures grown up last night to extract plasmids
  • Sent in the resulting plasmids for sequencing

Wednesday, 7/9/14

 Export Systems
  • Discovered that the reverse primer used in colony PCR last night had no binding site on the plasmids we had assembled (pTG001 constructs)
  • Miniprepped liquid cultures grown up last night to extract plasmids
    • Not all bacteria were able to be resuspended after pelleting, so resulting DNA yields for 6 of the 10 liquid cultures grown were very low (10-25 ng/uL)
Combinatorial Promoters
  • Analyzed sequencing results and grew more liquid cultures of the colonies containing properly assembled plasmids

Thursday, 7/10/14

Friday, 7/11/14

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