"

From 2013.igem.org

Revision as of 18:03, 22 September 2013 (view source) Smithpa (Talk | contribs) (→Notebook) ← Older edit		Latest revision as of 23:26, 22 September 2013 (view source) Hamptonite (Talk | contribs) (→E. coli Lab)
(23 intermediate revisions not shown)
Line 2:		Line 2:
	= Notebook =		= Notebook =
-	'''Methanococcus Lab'''
-	'''Tasks completed before 2013/02/20'''	+	== '''Methanococcus Lab''' ==
-	-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,	+	Instructor: ZHE LYU
-	anaerobic chamber, etc.	+
-	-Transferred and grew Methanococcus transformants with the geraniol synthase
-	gene encoded in the plasmid that was stored via glycerol stock from Summer
-	2012. Transformants containing only the pAW42 vector (control) were also
-	transferred and grown.
-	-Cell samples from the cultures listed above were harvested, resuspended in	+	'''February 2013'''
-	buffer and stored at -20C for running SDS in near future.	+
-		+
-	-Cell samples from cultures listed two above were harvested, flash-frozen and	+
-	stored at -80C formRNA extraction in the near future.	+
-	-Both liquid and solid anaerobic medium was prepared for future use. Refer to	+	-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,
-	Appendix of protocols, I Preparation of Complex Medium for Methanococci	+	anaerobic chamber, etc.
-	(McC).	+
-		+
-	'''2013/2/20'''	+
-		+
-	-Further preparation of media tubes via inoculation of Puromycin and	+
-	pressurization to 40 psi using H2/CO2 gas. Stored at -20C.	+
-		+
-	-Preparation of samples for SDS gel.	+
-		+
-	-Vector (control) samples made at 1x dilution and 5x dilution	+
-		+
-	-Geranyl Synthase (GS) plasmid samples made at 1x and 5x dilution	+
-		+
-	-An SDS gel was run using both dilutions of vector and GS samples, along with a	+
-	protein marker.	+
-		+
-	'''2013/2/26'''	+
-		+
-	-Four media tubes were treated with 0.1 mL Na2S solution each. Two of the tubes	+
-	were used for inoculation of GS transformant, and the other two were used for	+
-	inoculation of GS + AT (geraniol acetyltransferase) transformant.	+
-		+
-	-200x dilution of Puromycin solution was created and properly dispensed.	+
-		+
-	-SDS gel from 2013/2/20 was imaged. Gel image quality was lacking, so it was	+
-	decided to perform a second gel.	+
-		+
-	'''2013/2/27'''	+
-		+
-	-Reflecting upon first SDS gel, new sample dilutions were created in aim of	+
-	creating an easier to read gel. 2x dilution sample of GS was created and leftover	+
-	5x GS sample from first gel was also used.  4x, 6x and 8x dilutions of vector	+
-	samples were created, and leftover 5x dilution vector samples from first gel were	+
-	also used. Lastly, a protein marker ladder was used for size reference.	+
-		+
-	-Gel was run, stained, de-stained and imaged (see below). No evident expression	+
-	of GS protein. Next verification of this will be to run an mRNA extract for	+
-	conclusive evidence of GS gene being transcribed	+
-		+
-		+
-		+
-		+
-		+
-		+
-	[[[[[[[[[[[[[[[[[[[[Fig 1. ]]]]]]]]]]]]]]]]]]]	+
-		+
-		+
-		+
-		+
-		+
-		+
-		+
-	'''2013/03/05'''	+
-		+
-	-6 tubes of McC media created, added Na2S (0.1mL 2.5%) and Puromycin (0.5mL of	+
-	10x)	+
-		+
-	-Inoculated vector strain Methanococcus into two tubes and GS + AT into other two	+
-	tubes.	+
-		+
-		+
-	'''2013/03/06'''	+
-		+
-	-Solid McC media was autoclaved so gel would melt, so we could add Na2S and	+
-	Puromycin to all solid media bottles for plating. Puromycin was added to all but two	+
-	bottles. These two bottles will be used as control for Puromycin.	+
-		+
-	-GS and AT cell cultures were measured for their optical density (OD). A cultures OD	+
-	can tell us how well its growth and reproduction is. There is a direct correlation between	+
-	OD and cells/mL, so we will use this to determine how much we must dilute to achieve	+
-	around 150 cells/ml for optimal plating.  Appropriate dilutions were measured and	+
-	created.  These samples were added to solid media bottles is various quantities (0.3mL,	+
-	0.6mL and 0.9mL), allowed to rest on the agar media for half an hour, then placed	+
-	vertically into a 37C incubation room.	+
-		+
-	'''2013/03/19'''	+
-		+
-	-4 media tubes were prepared with 0.1 mL of 2.5% Na2S solution. These tubes	+
-	were then pressurized to 40 psi using H2/CO2 gas.	+
-		+
-	-Puromycin solution was prepared: 7.5mg of 200x Puromycin was added to 30	+
-	mL of de-ionized water and sparged in nitrogen gas. The 30 mL was then	+
-	transferred to 6 tubes, each with 5 mL of 100x Puromycin.	+
-		+
-	-6 solid media plates were prepared for future AT Methanococcus. The 6 bottles	+
-	were autoclaved, and treated with 0.1 mL 100x Puromycin and 0.2 mL 2.5%	+
-	Na2S solution each.	+
-		+
-	-1 GS (CK or control) colony, and 1 GS colony was slected and inoculated to	+
-	media containing 0.1 mL 2.5% Na2S. Note that these 2 colonies were not well	+
-	separated.  The 2 cultures were added to a 37C incubator.	+
-		+
-	'''2013/03/20'''	+
-		+
-	-200 mL liquid media was created, dispensed into 40 tubes, stoppered and capped,	+
-	pressurized to 15 psi using nitrogen and carbon dioxide, autoclaved and placed in the	+
-	anaerobic chamber.	+
-		+
-	-Frozen transformants of AT and GS+AT were placed into two properly treated media	+
-	tubes and placed into 37C incubator.	+
-		+
-	-Various appropriate dilutions of GS and AT cultures were created relative to OD	+
-	readings as described in the process from 2013/03/06.	+
-		+
-	-These dilutions were inoculated into 8 solid media bottles for plating, and stored at 37C	+
-	incubation room.	+
-		+
-	'''2013/03/26'''	+
-		+
-	-8 media tubes were treated with 0.1 mL of 2.5% Na2S solution, 0.05 mL of 100x	+
-	Puromycin solution and pressurized to 40 psi with H2 and CO2. Two of these tubes were	+
-	inoculated with 0.2 mL of AT transformant , while 2 other tubes were inoculated with 0.2	+
-	mL of AT+GS transformant.  The remaining 4 tubes were inoculated with colonies that	+
-	formed from the GS transformant after Puromycin purification.	+
-		+
-	-6 solid media bottles were autoclaved, treated with 0.2 mL Na2S and treated with 0.1	+
-	mL of Puromycin for future plating.	+
-		+
-	'''2013/03/27'''	+
-		+
-	-Re-pressurize H2/CO2 culture tubes to 40 psi	+
-		+
-	-Place 1 mL of 60% glycerol/40% media solution into 20 three-mL serum bottles. These	+
-	20 bottles were stoppered and capped for future frozen culture stocks.	+
-		+
-	-Check OD of cultures, and decided they were not sufficient enough for proper plating.	+
-		+
-	'''2013/03/28'''	+
-		+
-	-Re-pressurize H2/CO2 cultures tubes to 40 psi.	+
-		+
-	-Measure OD of cultures tubes hoping much better growth since 2013/03/27. The OD	+
-	measurements provided results satisfactory for plating.	+
-		+
-	-Various appropriate dilutions of AT and GS+AT cultures were created relative to OD	+
-	readings as described in the process from 2013/03/06.	+
-		+
-	-These diluted samples were inoculated into 6 solid media bottles for plating and plates	+
-	were placed into 37C incubation room.	+
-		+
-	-3 autoclaved glycerol stock bottles made 2013/03/27 were used to create frozen culture	+
-	stocks of biological replicates of GS. Namely, GS-1, GS-2 and GS-3 which were derived	+
-	from 3 different GS culture tubes. These 3 GS-inoculated glycerol stock bottles were	+
-	placed in -80C freezer.	+
-		+
-	'''2013/04/02'''	+
-		+
-	-400 mL of liquid media was created.	+
-		+
-	'''2013/04/03'''	+
-		+
-	-Liquid media created 2013/04/02 was dispensed into 40 tubes, stoppered and capped. All	+
-	tubes were pressurized to 15 psi using nitrogen and carbon dioxide then autoclaved.	+
-		+
-	-Agar was weighed out for 20 solid media bottles. Liquid media was then dispensed to	+
-	these solid media bottles in addition to the agar. These bottles were stoppered, capped,	+
-	pressurized and autoclaved like the previously mentioned 40 tubes.	+
-		+
-	-All tubes and bottles were moved to anaerobic chamber.	+
-		+
-	-Colonies were picked from an AT and AT+GS plate. Four colonies were taken from	+
-	each plate and inoculated into respective media tubes.	+
-		+
-	-Samples from the GS-1 and GS-2 frozen stock cultures were added to respective media	+
-	tubes to test if the previously created frozen stock cultures are actually viable.	+
-		+
-	-The 10 tubes including the AT colonies, AT+GS colonies, and frozen stock samples	+
-	were added to an incubator for growth.	+
-		+
-	'''2013/04/09'''	+
-		+
-	-1 mL of two different GS cultures (labeled GSF-1 and GSF-2) were inoculated in a 1 mL	+
-	media/glycerol solution and stored at -80C for future use as a frozen stock culture.	+
-	-6 agar bottles were autoclaved, treated with 0.1 mL of 100x Puromycin and 0.2 mL
-	2.5% Na2S. These bottles will be used for future plating.
-	-A test of Puromycin strength was started. 2 media tubes were used, one with 0.05 mL of	+	'''March 2013'''
-	Puromycin and 0.1 mL Na2S, while the other was not treated with anything (to serve as a	+
-	control). From there, 0.2 mL of Methanococcus was added to each tube, and the tubes	+
-	will be compared for Archaeal growth in the future.	+
-	'''2013/04/10'''	+	-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.
-	-Measure OD for AT and AT+GS cultures
-
-	-Various appropriate dilutions of AT and GS+AT cultures were created relative to OD
-	readings as described in the process from 2013/03/06.
-	-Make three plates for AT and three plates for AT+GS	+	'''April 2013'''
-	-2 technical replicates at 10^-6 dilution at 0.6mL and 0.9mL per plate (using the	+	-Further continuation of purification of GS, AT and GS+AT via picking colonies, creating sub-cultures and plating.
-	tube with the higher OD measurement).	+
-	-1 biological replicate at 10^-5 dilution at 0.6mL (lower of the tubes OD	+	-Test of Puromycin strength.
-	measurements).	+
-	-Create 9 glycerol stock serum bottles as described in 2013/03/27 for future frozen	+	-Creation of frozen stocks of purified GS, AT and GS+AT transformants.
-	stocks. 1 mLof glycerol was added, the bottles were then stoppered, capped, autoclaved	+
-	and moved into the anaerobic chamber.	+
-	'''2013/04/16'''
-	-The two media tubes containing Methanococcus from 2013/04/09 were measured for	+	'''June 2013'''
-	OD values. The Na2S + Puromycin containing media had on OD of 0.049A and the	+
-	control has an OD of 0.795. This data suggests that Puromycin is indeed still an effective	+
-	antibiotic.	+
-	-200 mL of liquid media was prepared.	+	-Extraction of Geraniol from extra-cellular and intra-cellular content of samples and preparation for GC/MS evaluation
-	'''2013/04/17'''	+	-Sequencing of pAW50-mCherry vector
-	-Liquid media created 2013/04/16 was dispensed, stoppered, capped, pressurized to 15	+	-Analysis of pAW50-mCherry sequence
-	psi using nitrogen and carbon dioxide, autoclaved and placed back into the anaerobic	+
-	chamber.	+
-	-1 plate of AT+GS and 1 plate of AT samples created 2013/04/10 were used for creating	+	-Transformation of pAW50-mCherry vector into Methanococcus
-	cultures.	+
-	-Four colonies from each plate were inoculated into respective media tubes for plating,	+	-Testing Fluorescence of pAW50-mCherry
-	and these plates were added to the incubator for growth.	+
-	2013/04/19	+	-Purification of pAW50-mCherry cultures via plating of transformants, and creating sub-cultures of colonies picked
-	-Colonies were picked from the plates created 2013/04/17 and inoculated into media	+
-	tubes. These tubes were added to a 37C incubator for growth. The success of these	+
-	colonies ends the purification process for all of our vectors.	+
		+	-Creation of frozen stocks of pAW50-mCherry in Methanococcus
-	[[[[[SUMMER SEMESTER]]]]]]]]]]]]]
-	'''2013/06/07'''	+	'''July 2013'''
-	'''Creation of Transformation Buffer (TB), Cysteine-HCL/DTT solution, and Mc media'''	+	-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).
-	Smith, Peyton. Rodriguez, Nicholas. Burroway, Brandon.	+	-Innovation of an adapter to allow the use of syringe needles on micropipettes.
-	-Creation of 100ml of transformation buffer in preparation of mcherry plasmid	+	-Expanded upon the original extraction protocol for higher efficiency of extraction of geraniol from Methanococcus cultures.
-	transformation.	+
-	-Creation of 20ml of Cysteine-HCL/DTT solution in preparation of mcherry plasmid	+	- GC/MS Evaluation and analysis (see results tab)
-	transformation	+
-		+
-	-Creation of 400ml of formate liquid media - to be dispensed in 40 tubes @ 5ml each and	+
-	20 agar bottles @ 10ml each	+
-		+
-	-All three of these were sparged for an appropriate time and placed inside the anaerobic	+
-	chamber.	+
-		+
-	'''2013/06/10-2013/06/14'''	+
-		+
-	'''Sequencing, Laundry and Transformation Preparation'''	+
-	Smith, Peyton. Rodriguez, Nicholas. Burroway, Brandon. Fetchko, Travis.
-	-mcherry plasmids were received from E. coli lab, and immediately sent to the sequencing
-	center on campus.
-	-All necessary buffers and solutions for methanococcus transformation were prepared.	+	'''August 2013'''
-	-Washing of all lab glassware.	+	-Revival and PCR of all GS and AT frozen stocks to confirm insert.
-		+
-	'''2013/06/17'''	+
-	'''Review of GC/MS Protocol, Extraction, GC/MS Preparations, Transformation Prep'''	+	--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation
-		+
-	Smith, Peyton. Fetchko, Travis. Hampton, Michael.	+
-	-Extraction of AT and wild type cultures for future GC/MS analysis of geraniol
-	production
-	-Creation of six S0001 culture tubes to be used as the recipient methanococci for mcherry	+	'''September 2013'''
-	transformation	+
-		+
-	-Preparation of geranyl acetate inoculant solution.	+
-	'''2013/06/18'''	+	-Transformation of V4 into Methanococcus
-	'''Creation of Future GC/MS-Evaluated Cultures, Extraction, GC/MS Prep'''	+	-Testing Fluorescence of V4
-		+
-	Smith, Peyton. Fetchko, Travis. Brandon, Burroway. Hampton, Michael.	+
-	-Inoculation of GS, AT, and AT+GS into 10 media tubes (2, 4, and 4, respectively)	+	-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked
-	-Geranyl acetate, originally created 2013/06/17, was added to two AT and two AT+GS	+	-Creation of frozen stocks of V4 in Methanococcus
-	tubes previously mentioned	+
-	-These 10 tubes will be used for GC/MS analysis of geraniol production.	+	-GC/MS Evaluation and analysis (see results tab)
-		+
-	-Creation of geraniol and geranyl acetate solution to be used when creating GC/MS	+
-	standards.	+
-	-Extraction of supernatant from GS and GS+AT cultures for future GC/MS analysis of	+	== '''E. coli Lab''' ==
-	geraniol production.	+
-	-Cell pellets from GS and GS+AT cultures were frozen in -20C for 1h and thawed before	+	Instructor: Rachit Jain
-	being resuspended	+
-		+
-	-Extraction of lysed cell pellets from GS and GS+AT cultures for future GC/MS analysis	+
-	of geraniol production	+
-	'''2013/06/19'''
-	'''Sequence analysis & Transformation Prep'''	+	'''February 2013'''
-	Smith, Peyton. Hampton, Michael.	+	-Training on PCR and heat shock transformation
-	-Analysis of mcherry plasmid sequence
-	-Final preparations for transformation	+	'''March 2013'''
-	'''2013/06/20'''	+	-PCR of GS gene
-	'''MCherry Plasmid Transformation into Methanococcus'''	+	-Cloning of GS gene into pAW-50.
-		+
-	Smith. Peyton. Hampton, Michael. Rhode, Benji. Fetchko, Travis. Rodriguez, Nicholas.	+
-	Burroway, Brandon.	+
-	-Transformation of mcherry plasmid into methanococcus was completed in two balch	+	-Transformation of pAW-50 GS into species XL1-Blue. Transformed species spread onto ampicillin plates
-	tubes according to protocol. These tubes were respectively labeled A and B.	+
-		+
-	-Transformation was also completed in parallel in a modified version of the protocol. The	+
-	majority of the transformation was completed inside the chamber in 1.5ml	+
-	microcentrifuge tubes that could be centrifuged inside the chamber. The volumes of	+
-	media and DNA were proportionally decreased according to the protocol. Four	+
-	microcentrifuge tubes were made and proceeded through the protocol until the first	+
-	addition of TB. Here, we combined the four microcentrifuge tubes into two to increase	+
-	the number of recipient cells as OD values are expected to decrease during the	+
-	transformation process. For TB and TB+PEG, one microcentrifuge tube received the	+
-	amount according to the protocol, whereas the other microcentrifuge tubes received 60%	+
-	the amount of TB and TB+PEG as the protocol indicates. These tubes were labeled A and	+
-	B, respectively. Due to microcentrifuge tube B’s inability to form a proper cell pellet, A	+
-	and B were combined into one balch tube, labeled AB, before being taken out of the	+
-	chamber where we added 1ml of media, pressurized, and added to the incubator	+
-	overnight.	+
-		+
-	'''2013/06/21'''	+
-	'''Plating of Transformants and Creation of Puromycin Enriched Transformant Cultures'''	+	-Verification of GS transformants via digestion and gel verification
-		+
-	Smith, Peyton.	+
-	-Serial dilutions of the transformants were created in dilutions of 10^-1, 10^-2 and 10^-3.
-	-For each of the three transformants, namely A, B and AB, plates were created using 1ml	+	'''April 2013'''
-	of inoculant for 10^-1 and 10^-2 and 2ml for 10^-3. A tenth plate was inoculated with	+
-	s0001 as a positive control.	+
-	-Puromycin was then added to all serial dilutions tubes so we may analyze fluorescence in	+	-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.
-	enriched cultures in parallel to growing colonies.  A puromycin enriched tube was also	+
-	created for a tube containing the pAW50 vector to serve as a negative control.	+
-	-All serial dilution tubes and negative control were added to the incubator, and all plates	+	-PCR and gel verification of mCherry gene
-	including positive control were added to the warm room.	+
-		+
-	'''2013/06/24'''	+
-	'''Transformation Results, GC/MS Sample Creation, GS, AT and GS+AT Inoculation/Revival'''	+	-Cloning of mCherry gene into pAW-50 vector
-	Smith, Peyton. Fetchko, Travis.	+	-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.
-	-OD of puromycin enriched cultures originally created 2013/06/21 were read. 10^-1 and	+	-XL1-Blue colonies picked and screened. Purification of pAW-50 mCherry from colonies, and stored in -20°C freezer.
-	10^-2 dilutions were between 0.6-0.9, but 10^-3 dilution was 0.2-0.4, so they were put	+
-	back into the incubator to continue growing.	+
-	-Plates of transformants of 10^-1 dilution are beginning to show small colonies.
-
-	-Hexane-extracted samples for GC/MS have finished drying, so final samples 100ul-200ul
-	were added to tubes and added to the iGEM box of GC/MS samples. These samples will
-	be analyzed for geraniol production in the GC/MS lab as soon as the lab becomes
-	available.
-	-GS, AT, and GS+AT cultures originally created 2013/06/18 for GC/MS analysis still	+	'''June 2013'''
-	show no signs of growth, so we decided to make new ones.	+
-		+
-	-The room temperature cultures used to create the cultures that were unsuccessful were	+
-	inoculated into 6 balch tubes, 3 with puromycin, 3 without.	+
-	-Glycerol stock samples were revived for each inoculant into 6 balch tubes, 2 tubes per	+	-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock
-	inoculant respectively. All 6 tubes were without puromycin These can ideally be	+
-	transferred into tubes with puromycin in the future.	+
-	-Note: glycerol stock GS-1 may be compromised due to prolonged thawing.	+	-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.
-		+
-	'''2013/06/25'''	+
-		+
-	'''Fluorescence Testing of MCherry, Creation of Media'''	+
-		+
-	Smith, Peyton. Fetchko, Travis. Rodriguez, Nicholas	+
-	-In E. coli lab: verification of successful expression of mcherry in methanococcus through	+	-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector
-	use of fluorometer. See Rachit for raw data.	+
-	-Prepared 400ml of formate media
-
-	'''2013/06/26'''
-
-	'''Picking MCherry Colonies & Dispensing Media'''
-	Smith, Peyton. Fetchko, Travis. Rodriguez, Nicholas.	+	'''July 2013'''
-	-Dispensed media originally created 2013/06/25 into 80 tubes which were stoppered,	+	-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.
-	sealed, pressurized to 15 psi using N2/CO2, and autoclaved.	+
-	-5 colonies from mcherry plates originally created 2013/06/21 were picked and inoculated	+	-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.
-	into 5 media tubes with 2.5% Na2S and puromycin. 1 colony was picked from sample B	+
-	10^-2, 1 colony was picked from sample C 10^-2, and 3 colonies were picked from	+
-	sample C 10^-1 and placed into respectively labeled tubes. These tubes were placed the	+
-	incubator.	+

---

Latest revision as of 23:26, 22 September 2013

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.