iGEM 2015 — Team William_and_Mary

# Team:William and Mary/Notebook

NOISE - W&M iGEM

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[W&M iGEM 2015](https://2015.igem.org/Team%3AWilliam_and_Mary)

* [Home](https://2015.igem.org/Team%3AWilliam_and_Mary)
* [Project Description](https://2015.igem.org/Team%3AWilliam_and_Mary/Description)
* [Parts](https://2015.igem.org/Team%3AWilliam_and_Mary#parts)
* [Results](https://2015.igem.org/Team%3AWilliam_and_Mary/Measurement)
* [Modeling](https://2015.igem.org/Team%3AWilliam_and_Mary/Modeling)
* [Interlab Study](https://2015.igem.org/Team%3AWilliam_and_Mary/Interlab_Study)
* [Collaboration](https://2015.igem.org/Team%3AWilliam_and_Mary/Collaborations)
* [Software](https://2015.igem.org/Team%3AWilliam_and_Mary/Software)
* [Human Practices](https://2015.igem.org/Team%3AWilliam_and_Mary/Practices)
* [References](https://2015.igem.org/Team%3AWilliam_and_Mary#ref)
* [Medal Criteria](https://2015.igem.org/Team%3AWilliam_and_Mary/Medal_Criteria)
* [Team](https://2015.igem.org/Team%3AWilliam_and_Mary/Team)

Our Notebook

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

![...](https://static.igem.org/mediawiki/2015/thumb/7/76/WMagenda.jpeg/450px-WMagenda.jpeg)

Important Protocols

Easy access to our most complex protocols.

[![...](https://static.igem.org/mediawiki/2015/c/c7/WMGibsonIconColor.png)](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AGibson)

[### Gibson Assembly](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AGibson)

For building constructs

[![...](https://static.igem.org/mediawiki/2015/7/7c/WMMicroscopeIconColor.png)](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AImaging)

[### Imaging](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AImaging)

Visualizing fluorescent bacteria

[![...](https://static.igem.org/mediawiki/2015/6/64/WMIntegrationIconColor.png)](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AIntegration)

[### Integration](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AIntegration)

Integrating onto the E. coli genome

[![...](https://static.igem.org/mediawiki/2015/1/18/WMElectroporationIconColor.png)](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AdCas9)

[### Repression](https://2015.igem.org/Team%3AWilliam_and_Mary/Protocols%3AdCas9)

Repressing with dCas9

Monthly Timeline

![...](https://static.igem.org/mediawiki/2015/thumb/b/b5/WMJune.jpg/800px-WMJune.jpg)
### June

Click for details.

![...](https://static.igem.org/mediawiki/2015/4/4a/WMJuly.png)
### July

Click for details.

![...](https://static.igem.org/mediawiki/2015/2/2f/WMAugust.jpg)
### August

Click for details.

![...](https://static.igem.org/mediawiki/2015/4/41/WMSeptember.jpg)
### September

Click for details.

# 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