"

From 2013.igem.org

Notebook

Methanococcus Lab

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]]]]]]]]]]]]]