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Team:UGA-Georgia/Notebook
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
[[[[[SUMMER SEMESTER]]]]]]]]]]]]]
2013/06/07
Creation of Transformation Buffer (TB), Cysteine-HCL/DTT solution, and Mc media
Smith, Peyton. Rodriguez, Nicholas. Burroway, Brandon.
-Creation of 100ml of transformation buffer in preparation of mcherry plasmid transformation.
-Creation of 20ml of Cysteine-HCL/DTT solution in preparation of mcherry plasmid 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.
-Washing of all lab glassware.
2013/06/17
Review of GC/MS Protocol, Extraction, GC/MS Preparations, Transformation Prep
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 transformation
-Preparation of geranyl acetate inoculant solution.
2013/06/18
Creation of Future GC/MS-Evaluated Cultures, Extraction, GC/MS Prep
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 tubes previously mentioned
-These 10 tubes will be used for GC/MS analysis of geraniol production.
-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 geraniol production.
-Cell pellets from GS and GS+AT cultures were frozen in -20C for 1h and thawed before 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
Smith, Peyton. Hampton, Michael.
-Analysis of mcherry plasmid sequence
-Final preparations for transformation
2013/06/20
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 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
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 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 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 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
Smith, Peyton. Fetchko, Travis.
-OD of puromycin enriched cultures originally created 2013/06/21 were read. 10^-1 and 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 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.
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 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.
-Dispensed media originally created 2013/06/25 into 80 tubes which were stoppered, 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 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.
2013/7/02-05
-Some of the AT, AT+GS and GS cultures inoculated on 6/24/13 came up and had O.D. taken by LZ. Those cultures were further subcultured.
2013/07/10
Creation of Sub-Cultures for Feeding Experiments
Smith, Peyton. Rodriguez, Nicholas.
-AT, GS, and AT+GS samples that were revived from frozen glycerol stocks were 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.
-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)
o AT+GS fed with geranyl acetate (GA) x2 (GA diluted in hexanes)
o AT+GS fed with GA x2 (GA diluted in ethanol)
2013/07/12
Extraction of AT and AT+GS and Creation of Killing/Inhibiting Experiment
Smith, Peyton. Rodriguez, Nicholas.
-Extraction protocol was completed for AT and AT+GS subcultures without any added feed that were originally created 2013/07/10. This extraction was completed for the sub- cultures’ supernatant.
-The fed cultures’ inability to grow in the same amount of time from biological replicates as those that did grow without any feed lead us to create another experiment, where we shall test Methanococcus’ reaction to the exposure of all possibilities that could have caused it to not grow.
-In this experiment we will test geranyl acetate (GA), geraniol, hexane, and ethanol’s affect on the growth/sustainability of Methanococcus.
-Six grown cultures with high OD values were used to create sub cultures for the inhibiting experiment. After inoculation of sub-cultures, the six original cultures were used for the killing experiment.
-List of samples and treatments: Inhibiting
o AT with hexane with respective negative control
o AT with ethanol with respective negative control
o AT x2 with GA with respective negative controls
o AT+GS x2 with geraniol with respective negative controls
-List of samples and treatments: killing
o AT with hexane
o AT with ethanol
o AT with GA x2
o AT+GS with geraniol x2
2013/07/15
Analysis of Killing/Inhibiting Experiment and Creation of Maximum Boundary Experiment
Smith, Peyton. Rodriguez, Nicholas.
-The following were observed from the killing/inhibiting experiment:
o Ethanol at the dilution used to introduce GA to a culture is enough to be both inhibiting to the growth of a culture and also enough to be lethal to a grown culture.
o Hexane at the dilution used to introduce GA to a culture has no effect on the growth or lethality of a culture.
o Geranyl Acetate (GA) introduced to a culture at a concentration of 10ng/5ml has no effect on the growth or lethality of a culture
o Geraniol inoculated cultures showed significantly higher OD readings in the growth cultures compared to their respective negative controls, but geraniol inoculated cultures with initially high OD values, had no change. Geraniol may be beneficial to the growth of methanococcus, but this observation is inconclusive. Future experiments will be conducted to further test this hypothesis.
-Since cultures grew well in 10ng/5ml conentration of GA and geraniol, we decided to begin a new experiment to test the upper boundary of how much GA and geraniol a 5ml methanococcus culture could handle, also as a test to determine methanococcus’ highest possible production yield, at least in its current condition.
-Sub-cultures of AT and AT+GS were created and inoculated with GA. For these 2 samples, 2 treatments were created, namely 10ng/5ml and 1000ng/5ml, and 1 replicate was created for al. These samples were placed into the incubator for growth
-Note: for inoculation, we ran out of GA solution, so one of the tubes which was labeled to have 1000ng of GA added to it actually received <500ng GA, but we did not mark which one it was.
-Innovation of an adapter to allow the use of syringe needles on micropipettes.
2013/07/16
Creation of Formate Media, 100ml Cultures, and Inoculation of 100ml Cultures
Smith, Peyton. Rodriguez, Nicholas. Hampton, Michael.
-After some calculations taking into account the lipid biosynthesis pathway that geraniol is created from, it is very unlikely that 5ml cultures are able to produce the 2ng/ul of geraniol that is necessary for detection in GC/MS. We decided to create three 100ml cultures of AT, GS, and AT+GS so that we may break the 2ng/ul detection limit.
-300ml x2 of media was created, pressurized, autoclaved and placed into the chamber to later be dispensed into three large culture bottles and 60 media tubes.
-The three 100ml cultures were inoculated with 5ml of our highest-OD culture of AT, GS, and AT+GS without any feed that was originally created 2013/07/10. This inoculation process should be completed through use of a vacutainer needle. Note: repressurize the 5ml culture tubes before inoculating via vacutainer needle!
-These three 100ml culture bottles were placed into metal cylinders (barrels) and placed into the large incubator.
2013/07/18
Further Investigation of Maximum Boundary Experiment and GC/MS Booking
Smith, Peyton. Rodriguez, Nicholas. Burroway, Brandon. Rahul.
-OD values were read of the feeding experiment created 2013/07/15. One of the tubes from the experiment somehow was exposed to oxygen, so the culture was pink, dead and not usable. All of the remaining tubes which had 10ng of GA grew with high OD values. All of the tubes with 1000ng of GA did not show any growth except for one. One tube labeled to have added 1000ng of GA did grow, could this have been the one that actually received <500ng? The result it this is inconclusive because we did not mark which tube received <500ng GA
-New feeding experiments were created to further test the upper limit of GA/geraniol tolerance in methanococcus. 12 tubes were taken to create a similar experiment using 2 samples, 3 treatments, and 1 replicate. The samples were AT and AT+GS, the treatments were 100ng, 500ng, and 1000ng of GA, and 1 replicate of each to ensure results. Note: these tubes contain puromycin.
-OD values of the three 100ml cultures were taken, none of which were high enough to begin extraction, but AT and AT+GS were moved to room temperature, and GS was added back to the incubator.
-We visited Dr. Dennis Phillips to discuss GC/MS. We booked an official GC/MS appointment to test for geraniol on Tuesday, 2013/07/23
2013/07/19
Modification of Extraction Protocol, Extraction via Modified Protocol, Creation of GC/MS Standards, Extraction Efficiency Experiment.
Smith, Peyton. Burroway, Brandon.
-Research was done looking into the solubility of geraniol into hexane, and the consequential efficiency of the extraction procedure. See: Coriander_oil.pdf and http://www.nbb.cornell.edu/neurobio/ragusolab/Floral%20Scent%20primer.html
-We found that geraniol’s solubility in hexane is 1ug/ml, when we’ve been using 15ml of hexane to extract ~10ng of potential geraniol. Also, we found that solubility is increased through the use of a 9:1 solution of hexane : acetone. We decided to modify the extraction protocol in accordance to these findings to increase extraction efficiency and efficiency of sample drying.
-9:1 hexane/acetone solution was created and sealed for future use
-GC/MS geraniol standards were created. These standards include concentrations of 84ng/ul, 42 ng/ul, 8.4ng/ul and 0.84ng/ul
-OD values were read for the three 100ml cultures, and they were within enough range to begin extraction.
-Modified version of extraction protocol (see below) was completed for the extraction of the three 100ml culture of AT, GS and AT+GS. It is not known for sure whether or not geraniol created by methanococcus is excreted from the cell. So, extraction protocol for the cellular content (see below) was completed for the cells pelleted in the extraction protocol.
-Extraction protocol was completed for an extraction efficiency experiment using anerobic water and 840ng geraniol.
-Drying was completed down to 10ul for the supernatent extraction from the 100ml cultures of AT, GS and AT+GS and were added to GC/MS sample bottles.
-Cellular content extraction for all three samples and extraction efficiency had not finished drying and will be completed Monday.
-Innovation of a creative device to increase drying efficiency of multiple samples by 100%.
2013/07/22
Extraction of Feeding-Experiments & Final Preparation for GC/MS Smith, Peyton. Rodriguez, Nicholas.
-Drying process was completed for the remaining samples that were extracted 2013/07/19 including cell extract of all three 100ml cultures and the extraction efficiency experiment.
-Extraction was completed via modified protocol for four feeding-experiment cultures originally created 2013/07/18. These four cultures were single replicates AT and AT+GS which had 500ng and 1000ng GA treatments.
-All extraction samples were prepared for GC/MS and final gathering of materials/preparations were done.
2013/07/23
GC/MS Evaluation and Analysis
Smith, Peyton. Fetchko, Travis. Hampton, Michael. Westbrook, Marquise.
-GC/MS evaluation and analysis was completed for over 30 samples.
-See all raw data (via LZ or Peyton)
-Three culture samples gave positive results for production of geraniol. (see below)
-Modified extraction protocol shows ~65% efficiency, not differing much from the original extraction protocol.
[[[[[[[[[[[[[Fig. 3 Geraniol Standard (sample 32, approx. 8.4ng/ul)]]]]]]]]]]]]]]]]]
[[[[[[[[[[[[Fig. 4 AT or WT Supernatant (sample 6) ]]]]]]]]]]]]]]]]]]]]]]]]
[[[[[[[[[[[[[[[Fig. 5 Geraniol matched against database of mass spec entries peak at 7.81min (sample 6)]]]]]]]]]]
[[[[[[[[[Fig. 6 GS or AT+GS S/C (sample 7)]]]]]]]]]]]]]
[[[[[[[[[[Fig. 7 AT(0.46) Supernatant (sample 12) ]]]]]]]]]]]]]
2013/07/30
GC/MS Analysis and Revival of Frozen Stocks for Future GC/MS Trials
Smith, Peyton. Hampton, Michael. Rodriguez, Nicholas.
-Analysis of GC/MS results
-Revived 4 AT, 4 GS, and 4 AT+GS samples from frozen glycerol stock, all to be evaluated by GC/MS for geraniol production
-2 sub-cultures of wild type s0001 were created from 1 room temperature stock
-Subcultures of the 5 mCherry colonies picked were created to later be made into frozen stocks. These 5 subcultures were labeled c1-5, respectively.
-Note: Samples GS-4, GS+AT-8, and AT-12 were inoculated with 0.2ml from frozen glycerol stocks due to prolonged thawing.
[[[[[[[[[FALL SEMESTER]]]]]]]]]]]]]]
2013/08/06
Geraniol Extractions
Smith, Peyton
-Take OD values of the 19 cultures (originally created 2013/07/31)
-Complete extraction protocol for all cultures with OD value greater than 0.5
o GS: 15ml hexanes, supernatant and cell extract
o AT and AT+GS: 15ml hexanes, supernatant
o Wild Type: 15ml hexanes, supernatant and cell extract
o Evaporate to 200ul and 50ul for supernatant and cell extract, respectively
-Re-inoculate not yet grown cultures
o pAW50-mCherry: use original parent strains
o AT and AT+GS: use grown replicate strains.
-Wash GC/MS bottles with ethanol
2013/08/09
Geraniol Extractions and mCherry Frozen Stock
Smith, Peyton
-Take OD values for cultures that were re-inoculated on 2013/08/06
-Complete extraction protocol for all remaining cultures
-Create pAW-50 mCherry-C2 frozen stock.
2013/08/12
GC/MS Final Preparations and mCherry Frozen Stocks
Smith, Peyton
-Finish all extractions and place all samples into GC/MS sample bottles
-Create frozen stocks of pAW-50 mCherry C1 and C5
-Assemble and autoclave anaerobic glass syringes
2013/08/14
GC/MS Evaluation and Analysis
-10 samples were run using the method described in the first GC/MS evaluation
-No positive results came from these 10 samples.
-The positive samples from the first GC/MS evaluation were run, and still showed presence of geraniol.
-See raw data (via LZ)
-Our hypothesis is that we did not receive any positive results from the second round of GC/MS because we restricted ourselves to extracting from only 1 GS frozen stock which was derived from 1 colony during the purification process. In comparison, the first GC/MS trials used at least 2 GS frozen stocks/colonies. We have decided to begin a final geraniol production centred experiment. We will revive all frozen stocks/colonies of GS and AT, verify for insert via PCR and complete extraction. We hope by including all of the possible colony derivatives, we can reach conclusive results and determine which frozen stock produced geraniol in the first GC/MS trials.
2013/08/21
Inoculation of Samples for Future GC/MS Evaluations
Smith, Peyton
-GS-1, GS-3, and GSF samples were all revived with respective biological replicates.
-AT-1, AT-3, and AT-4 samples were all revived.
-Room-temp wild type samples were inoculated to create 3 samples for future extraction
2013/08/23
PCR of GS and AT Cultures to Confirm Insert
Smith, Peyton. Westbrook, Marquise. Hampton, Michael.
-OD values of the cultures inoculated on 2013/08/21 were taken.
-PCR was completed for all the aforementioned GS and AT samples. The following is the gel from the PCR confirming that some of the GS cultures indeed contain the GS gene and some of the AT samples contain the AT gene.
[[[[[[[[[[[[[[[Fig. PCR gel of GS and AT cultures from LZ]]]]]]]]]]]]]]]]
2013/08/26
Extraction of Geraniol from PCR-verified GS Cultures and Wild Type Cultures
Smith, Peyton
-The original extraction protocol was completed for all GS cultures and the three wild type cultures originally created 2013/08/21
-The 15ml organic solvent from extraction of geraniol was split into two 7.5ml containers to dry. One 7.5ml sample was allowed to dry naturally under the fume hood, and the other 7.5ml sample was dried under a stream of N2 gas.
2013/09/02
GC/MS Sample Preparation, and Preparation of V4 Transformation
Smith, Peyton
-All samples for GC/MS evaluation are prepared, and we have booked a time to work in the GC/MS lab.
-All necessarily materials were prepared for the transformation of V4 into methanococcus.
2013/09/03
Transformation of V4 into Methanococcus
.
2013/09/04
Plating of V4 Transformants, Creation of Puromycin-Enriched Transformant Cultures
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2013/09/05
Testing of Fluorescence of Puromycin-Enriched V4 Transformants
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2013/09/06
GC/MS Evaluation and Analysis
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2013/09/09
Picking V4 Colonies
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2013/09/11
Creation of Permanent Frozen Stocks of V4 in Methanococcus
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E. coli Lab
Instructor: Rachit Jain
