iGEM 2013 — Team UGA-Georgia

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# Team:UGA-Georgia/Notebook

### From 2013.igem.org

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Georgia iGEM 2013

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* [Home](https://2013.igem.org/Team%3AUGA-Georgia)
* [Plan](https://2013.igem.org/Team%3AUGA-Georgia/Plan)
* [Protocols](https://2013.igem.org/Team%3AUGA-Georgia/Protocol)
* Notebook
  + [E. coli](https://2013.igem.org/Team%3AUGA-Georgia/NotebookE.coli)
  + [Methanococcus](https://2013.igem.org/Team%3AUGA-Georgia/NotebookMethanococcus)
* [Results](https://2013.igem.org/Team%3AUGA-Georgia/Results)
* [Parts](https://2013.igem.org/Team%3AUGA-Georgia/Parts)
* [Safety](https://2013.igem.org/Team%3AUGA-Georgia/Safety)
* Outreach
  + [Engineering Seminar](https://2013.igem.org/Team%3AUGA-Georgia/Outreach/EngineeringSeminar)
  + [Clarke Middle School](https://2013.igem.org/Team%3AUGA-Georgia/Outreach/ClarkeMiddleSchool)
* [Human Practice](https://2013.igem.org/Team%3AUGA-Georgia/humanpractice)
* The Team
  + [Meet The Team](https://2013.igem.org/Team%3AUGA-Georgia/Team)
  + [Official Team Profile](https://igem.org/Team.cgi)
* [Acknowledgements](https://2013.igem.org/Team%3AUGA-Georgia/Acknowledgements)

# Notebook

## **Methanococcus Lab**

Instructor: ZHE LYU

**February 2013**

-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,
anaerobic chamber, etc.

**March 2013**

-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.

**April 2013**

-Further continuation of purification of GS, AT and GS+AT via picking colonies, creating sub-cultures and plating.

-Test of Puromycin strength.

-Creation of frozen stocks of purified GS, AT and GS+AT transformants.

**June 2013**

-Extraction of Geraniol from extra-cellular and intra-cellular content of samples and preparation for GC/MS evaluation

-Sequencing of pAW50-mCherry vector

-Analysis of pAW50-mCherry sequence

-Transformation of pAW50-mCherry vector into Methanococcus

-Testing Fluorescence of pAW50-mCherry

-Purification of pAW50-mCherry cultures via plating of transformants, and creating sub-cultures of colonies picked

-Creation of frozen stocks of pAW50-mCherry in Methanococcus

**July 2013**

-Creation and analysis of the "killing & inhibiting" experiment where we test the maximum amount of product a 5ml culture of Methanococcus can tolerate in cultures with high OD (killing of grown cells) and low OD (inhibiting of growth).

-Innovation of an adapter to allow the use of syringe needles on micropipettes.

-Expanded upon the original extraction protocol for higher efficiency of extraction of geraniol from Methanococcus cultures.

- GC/MS Evaluation and analysis (see results tab)

**August 2013**

-Revival and PCR of all GS and AT frozen stocks to confirm insert.

--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation

**September 2013**

-Transformation of V4 into Methanococcus

-Testing Fluorescence of V4

-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked

-Creation of frozen stocks of V4 in Methanococcus

-GC/MS Evaluation and analysis (see results tab)

## **E. coli Lab**

Instructor: Rachit Jain

**February 2013**

-Training on PCR and heat shock transformation

**March 2013**

-PCR of GS gene

-Cloning of GS gene into pAW-50.

-Transformation of pAW-50 GS into species XL1-Blue. Transformed species spread onto ampicillin plates

-Verification of GS transformants via digestion and gel verification

**April 2013**

-Colonies for GS, AT, and GS+AT were selected from Methanococcus and corresponding genes were extracted. PCR and gel verification of GS, AT, and GS+AT genes. Results concluded successful in locating vectors from Methanococcus.

-PCR and gel verification of mCherry gene

-Cloning of mCherry gene into pAW-50 vector

-Verification of cloning via digestion and gel verification. After positive verification, ligation of digested products and heat shock transformation into XL1-Blue. XL1-Blue transformants spread onto ampicillin plates.

-XL1-Blue colonies picked and screened. Purification of pAW-50 mCherry from colonies, and stored in -20°C freezer.

**June 2013**

-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock

-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.

-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector

**July 2013**

-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.

-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.

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