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Team:UGA-Georgia/Notebook
From 2013.igem.org
Notebook
Methanococcus Lab
Instructor: ZHE LYU
Tasks completed before 2013/02/20
-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 2012. 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 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 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.
2013/04/19 -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]]]]]]]]]]]]]
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
E. coli Lab
Instructor: Rachit Jain
