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Revision as of 21:41, 19 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.

...

Important Protocols

Easy access to our most complex protocols.

...

Gibson Assembly

For building constructs

...

Imaging

Visualizing fluorescent bacteria

...

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