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Team:UGA-Georgia/NotebookMethanococcus
From 2013.igem.org
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.
