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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.

@@ Line 5: / Line 5: @@
 == '''Methanococcus Lab''' ==
+Instructor: ZHE LYU
-'''Tasks completed before 2013/02/20'''
+'''February 2013'''
 -Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,
 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
-. Transformants containing only the pAW42 vector (control) were also
-transferred and grown.
--Cell samples from the cultures listed above were harvested, resuspended in
-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
-Appendix of protocols, I Preparation of Complex Medium for Methanococci
-(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
-x 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
-x)
--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,
-.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
-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
-.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
-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'''
--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
--2 technical replicates at 10^-6 dilution at 0.6mL and 0.9mL per plate (using the
-tube with the higher OD measurement).
--1 biological replicate at 10^-5 dilution at 0.6mL (lower of the tubes OD
-measurements).
--Create 9 glycerol stock serum bottles as described in 2013/03/27 for future frozen
-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
-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.
-'''2013/04/17'''
--Liquid media created 2013/04/16 was dispensed, stoppered, capped, pressurized to 15
-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
-cultures.
--Four colonies from each plate were inoculated into respective media tubes for plating,
-and these plates were added to the incubator for growth.
-/04/19
--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.
-[[[[[SUMMER SEMESTER]]]]]]]]]]]]]
+'''March 2013'''
+-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.
-'''2013/06/07'''
-'''Creation of Transformation Buffer (TB), Cysteine-HCL/DTT solution, and Mc media'''
+'''April 2013'''
-Smith, Peyton. Rodriguez, Nicholas. Burroway, Brandon.
+-Further continuation of purification of GS, AT and GS+AT via picking colonies, creating sub-cultures and plating.
--Creation of 100ml of transformation buffer in preparation of mcherry plasmid
+-Test of Puromycin strength.
-transformation.
--Creation of 20ml of Cysteine-HCL/DTT solution in preparation of mcherry plasmid
+-Creation of frozen stocks of purified GS, AT and GS+AT transformants.
-transformation
--Creation of 400ml of formate liquid media - to be dispensed in 40 tubes @ 5ml each and
-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
+'''June 2013'''
-center on campus.
--All necessary buffers and solutions for methanococcus transformation were prepared.
+-Extraction of Geraniol from extra-cellular and intra-cellular content of samples and preparation for GC/MS evaluation
--Washing of all lab glassware.
+-Sequencing of pAW50-mCherry vector
-'''2013/06/17'''
-'''Review of GC/MS Protocol, Extraction, GC/MS Preparations, Transformation Prep'''
+-Analysis of pAW50-mCherry sequence
-Smith, Peyton. Fetchko, Travis. Hampton, Michael.
--Extraction of AT and wild type cultures for future GC/MS analysis of geraniol
+-Transformation of pAW50-mCherry vector into Methanococcus
-production
--Creation of six S0001 culture tubes to be used as the recipient methanococci for mcherry
+-Testing Fluorescence of pAW50-mCherry
-transformation
--Preparation of geranyl acetate inoculant solution.
-'''2013/06/18'''
+-Purification of pAW50-mCherry cultures via plating of transformants, and creating sub-cultures of colonies picked
-'''Creation of Future GC/MS-Evaluated Cultures, Extraction, GC/MS Prep'''
+-Creation of frozen stocks of pAW50-mCherry in Methanococcus
-Smith, Peyton. Fetchko, Travis. Brandon, Burroway. Hampton, Michael.
--Inoculation of GS, AT, and AT+GS into 10 media tubes (2, 4, and 4, respectively)
--Geranyl acetate, originally created 2013/06/17, was added to two AT and two AT+GS
+'''July 2013'''
-tubes previously mentioned
--These 10 tubes will be used for GC/MS analysis of geraniol production.
+-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).
--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
+-Innovation of an adapter to allow the use of syringe needles on micropipettes.
-geraniol production.
--Cell pellets from GS and GS+AT cultures were frozen in -20C for 1h and thawed before
+-Expanded upon the original extraction protocol for higher efficiency of extraction of geraniol from Methanococcus cultures.
-being resuspended
--Extraction of lysed cell pellets from GS and GS+AT cultures for future GC/MS analysis
-of geraniol production
-'''2013/06/19'''
+- GC/MS Evaluation and analysis (see results tab)
-'''Sequence analysis & Transformation Prep'''
-Smith, Peyton. Hampton, Michael.
--Analysis of mcherry plasmid sequence
+'''August 2013'''
--Final preparations for transformation
+-Revival and PCR of all GS and AT frozen stocks to confirm insert.
-'''2013/06/20'''
+--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation
-'''MCherry Plasmid Transformation into Methanococcus'''
-Smith. Peyton. Hampton, Michael. Rhode, Benji. Fetchko, Travis. Rodriguez, Nicholas.
-Burroway, Brandon.
--Transformation of mcherry plasmid into methanococcus was completed in two balch
+'''September 2013'''
-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'''
+-Transformation of V4 into Methanococcus
-Smith, Peyton.
--Serial dilutions of the transformants were created in dilutions of 10^-1, 10^-2 and 10^-3.
+-Testing Fluorescence of V4
--For each of the three transformants, namely A, B and AB, plates were created using 1ml
+-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked
-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
+-Creation of frozen stocks of V4 in 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
+-GC/MS Evaluation and analysis (see results tab)
-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'''
+== '''E. coli Lab''' ==
-Smith, Peyton. Fetchko, Travis.
+Instructor: Rachit Jain
--OD of puromycin enriched cultures originally created 2013/06/21 were read. 10^-1 and
-^-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.
+'''February 2013'''
--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
+-Training on PCR and heat shock transformation
-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
-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.
+'''March 2013'''
-'''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
+-PCR of GS gene
-use of fluorometer. See Rachit for raw data.
--Prepared 400ml of formate media
+-Cloning of GS gene into pAW-50.
-'''2013/06/26'''
-'''Picking MCherry Colonies & Dispensing Media'''
-Smith, Peyton. Fetchko, Travis. Rodriguez, Nicholas.
+-Transformation of pAW-50 GS into species XL1-Blue. Transformed species spread onto ampicillin plates
--Dispensed media originally created 2013/06/25 into 80 tubes which were stoppered,
+-Verification of GS transformants via digestion and gel verification
-sealed, pressurized to 15 psi using N2/CO2, and autoclaved.
--5 colonies from mcherry plates originally created 2013/06/21 were picked and inoculated
-into 5 media tubes with 2.5% Na2S and puromycin. 1 colony was picked from sample B
-^-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.
+'''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.
-'''2013/7/02-05'''
+'''June 2013'''
--Some of the AT, AT+GS and GS cultures inoculated on 6/24/13 came up and had O.D. taken by
+-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock
-LZ. Those cultures were further subcultured.
-'''2013/07/10'''
+-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.
-'''Creation of Sub-Cultures for Feeding Experiments'''
+-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector
-Smith, Peyton. Rodriguez, Nicholas.
--AT, GS, and AT+GS samples that were revived from frozen glycerol stocks were
+'''July 2013'''
-observed with high OD values.
--Sub cultures were created of these samples so that we may use them for future feeding
-experiments when measuring geraniol production.
--These sub cultures were placed in the incubator overnight.
+-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.
--List of sub-cultures:
-o GS without any feed x2 (one was inoculated from room-temp culture, and the
-other was direct inoculation from frozen stock)
-o AT without any feed x2 (derived from room temp cultures)
-o AT fed with geranyl acetate (GA) x2 (GA diluted in hexanes)
-o AT fed with GA x2 (GA diluted in ethanol)
-o AT+GS without any feed x2 (derived from room temp cultures)
+-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.
-o AT+GS fed with geranyl acetate (GA) x2 (GA diluted in hexanes)
-o AT+GS fed with GA x2 (GA diluted in ethanol)

Team:UGA-Georgia/Notebook

From 2013.igem.org

Latest revision as of 23:26, 22 September 2013

Notebook

Methanococcus Lab

E. coli Lab