Difference between revisions of "Team:William and Mary/Notebook"

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                                               <a href = "https://2015.igem.org/Team:William_and_Mary/Protocols:dCas9"><img alt="..." src="https://static.igem.org/mediawiki/2015/1/18/WMElectroporationIconColor.png" height= "100" width= "120"/><a>
 
                                               <a href = "https://2015.igem.org/Team:William_and_Mary/Protocols:dCas9"><img alt="..." src="https://static.igem.org/mediawiki/2015/1/18/WMElectroporationIconColor.png" height= "100" width= "120"/><a>
 
                                           </div>
 
                                           </div>
                                           <a href = "https://2015.igem.org/Team:William_and_Mary/Protocols:dCas9"><h3>Promoter Repression</h3></a>
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                                           <a href = "https://2015.igem.org/Team:William_and_Mary/Protocols:dCas9"><h3>Repression</h3></a>
 
                                           <p>Repressing with dCas9</p>
 
                                           <p>Repressing with dCas9</p>
 
                                       </div>
 
                                       </div>

Revision as of 04:23, 16 November 2015

NOISE - W&M iGEM

Our Notebook

On this page you can view the work we did each month, from June through September. You can also find our protocols.

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Important Protocols

Easy access to our most complex protocols.

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Gibson Assembly

For building constructs

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Imaging

Visualizing fluorescent bacteria

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Integration

Integrating onto the E. coli genome

Repression

Repressing with dCas9

June

Week 1 (6/1-6/7)

  • Resuspended all parts from the kit
  • Designed Gibson assemblies to put various fluorescent proteins under the control of the same promoter, to get noise measurements
  • Began performing Gibson PCRs

Week 2 (6/8-6/14)

  • Troubleshot Gibson PCRs
  • Attempted first Gibson assembly
  • Designed Gibson assemblies to put together different fluorescent proteins, each under the control of the same promoter
  • Attempted to Gibson assemble YFP + CFP under control of R0010

Week 3 (6/15-6/21)

  • Re-attempted Gibson assembly to make a double fluorescent construct with proteins under R0010
  • Began assembling parts for the Interlab Study
  • Received gBlocks to assemble dCas9 and some preliminary gRNAs
  • Began assembling dCas9 and gRNAs
  • Began learning how to use confocal microscopy
  • Began assembling different promoters with our fluorescent constructs (to eventually collect noise data for each promoter)
  • Designed and started the assembly process to make KanR (necessary to integrate parts onto the E. coli genome)

Week 4 (6/22-6/28)

  • Many of our past assemblies (dCas9, double fluorescent constructs, etc) failed. We continued attempts at these assemblies.

July

Week 1 (6/29-7/5)

  • We have assembled our gRNAs and dCas9!
  • We ordered primers to integrate onto the E. coli genome
  • Designed assembly to put a KanR operon after a fluorescent construct to integrate
  • Designed a better way to assemble double fluorescent constructs
  • Designed a way to make a TetR operon to integrate two different things
  • Made electrocompetent cells containing our dCas9 operon
  • Assembled a double fluorescent construct! CFP and YFP under R0010
  • Attempted our first double electroporation-transformation to test our dCas9 and gRNAs

Week 2 (7/6-7/12)

  • Began assembling CFP and YFP operons with each promoter that we wanted to test
  • Confirmed that dCas9 and our gRNA design works
  • Assembled the CFP under R0010 with KanR, so that it can be PCR’d with the integration PCRs, and integrated
  • Attempted to make TetR operon

Week 3 (7/13-7/19)

  • Attempted to test our integration protocol, by integrating KanR onto the E. coli genome
  • Continued attempting to construct Interlab Parts
  • Worked on our collaboration with UGA
  • Designed gRNAs for other promoters to create a suite of repression parts
  • Continued assembling CFP and YFP operons with promoters of interest
  • Continued attempts to make TetR operon

Week 4 (7/20-7/26)

  • Began testing integrator cassette part
  • Continued working on Interlab Study
  • Continued assembling CFP and YFP operons
  • Continued attempts to make TetR operon

Week 5 (7/26-8/2)

  • Ordered gBlocks for Interlab Study
  • Continued working on assembling all gRNA parts
  • Continued assembling CFP and YFP operons
  • Imaged our single integrated fluorescent CFP (under R0010)
  • Continued attempts to make TetR operon

August

Week 1 (8/3-8/9)

  • Continued assembling CFP and YFP operons
  • Began assembling CFP operons with KanR (to integrate)
  • Continued attempts to make TetR operon

8/10-8/23: School mandates that we leave

  • During this time, we learned that TetR had been mislabelled, and the part we were actually looking to assemble to confer tetracycline resistance was “TetA”
  • We designed PCRs to assemble TetA

Week 4 (8/24-8/30)

  • Continued working on Interlab Study
  • Began assembling YFP operons with TetA (to integrate)
  • Began attempts to integrate YFP operons with TetA for which there was a corresponding CFP operon with KanR

September

Week 1 (8/31-9/6)

  • Integrated CFP operons with KanR
  • Integrated YFP operons with TetA into E. coli containing integrated CFPs

Week 2 (9/7-9/13)

  • Continued attempts to integrate YFPs into E. coli containing integrated CFPs
  • Imaged successful double integrations for R0010, R0011, R0051
  • Analyzed data from double integrations