Team:William and Mary/Notebook
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. Detail1 Detail2 Detail3 Detail4 Week 1 (7/1-7/7)
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 (7/8-7/14)
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 (7/15-7/21)
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 (7/22-7/28)
Many of our past assemblies (dCas9, double fluorescent constructs, etc) failed. We continued attempts at these assemblies.
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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
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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
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