Team:UGA-Georgia/NotebookMethanococcus

From 2013.igem.org

(Difference between revisions)

Revision as of 19:10, 23 September 2013

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)

@@ Line 1: / Line 1: @@
 <div id="header">{{Template:Team:UGA-Georgia/Templates/Header}}</div>
-= Notebook =
+==Methanococcus Lab ==
-== '''Methanococcus Lab''' ==
 Instructor: ZHE LYU
@@ Line 75: / Line 73: @@
 -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.