Team:UGA-Georgia/Notebook

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= Notebook =
= Notebook =
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'''Methanococcus Lab'''
 
-
'''Tasks completed before 2013/02/20'''  
+
== '''Methanococcus Lab''' ==
 +
 
 +
Instructor: ZHE LYU
 +
 
 +
 
 +
'''February 2013'''
-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,  
-Training on anaerobic skills, i.e. use of anaerobic glassware, gassing chamber,  
anaerobic chamber, etc.  
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
+
'''March 2013'''
-
buffer and stored at -20C for running SDS in near future.
+
 
-
+
-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.
-
-Cell samples from cultures listed two above were harvested, flash-frozen and
+
 
-
stored at -80C formRNA extraction in the near future.  
+
 
 +
'''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)
-
-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
+
'''August 2013'''
-
pressurization to 40 psi using H2/CO2 gas. Stored at -20C.
+
-
-Preparation of samples for SDS gel.
+
-Revival and PCR of all GS and AT frozen stocks to confirm insert.
-
+
-
-Vector (control) samples made at 1x dilution and 5x dilution
+
-
-Geranyl Synthase (GS) plasmid samples made at 1x and 5x dilution
+
--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation
-
-An SDS gel was run using both dilutions of vector and GS samples, along with a
 
-
protein marker. 
 
-
'''2013/2/26'''  
+
'''September 2013'''
-
-Four media tubes were treated with 0.1 mL Na2S solution each. Two of the tubes
+
-Transformation of V4 into Methanococcus
-
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
+
-Testing Fluorescence of V4
-
decided to perform a second gel.
+
-
'''2013/2/27'''
+
-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked
-
-Reflecting upon first SDS gel, new sample dilutions were created in aim of  
+
-Creation of frozen stocks of V4 in Methanococcus
-
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
+
-GC/MS Evaluation and analysis (see results tab)
-
of GS protein. Next verification of this will be to run an mRNA extract for
+
-
conclusive evidence of GS gene being transcribed 
+
-
[[[[[[[[[[[[[[[[[[[[Fig 1. ]]]]]]]]]]]]]]]]]]]
+
== '''E. coli Lab''' ==
 +
Instructor: Rachit Jain
-
'''2013/03/05'''
 
-
-6 tubes of McC media created, added Na2S (0.1mL 2.5%) and Puromycin (0.5mL of
+
'''February 2013'''
-
10x)
+
-
-Inoculated vector strain Methanococcus into two tubes and GS + AT into other two
+
-Training on PCR and heat shock transformation
-
tubes. 
+
-
2013/03/06
+
'''March 2013'''
-
-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
+
-PCR of GS gene
-
-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
+
-Cloning of GS gene into pAW-50.
-
-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
+
-Transformation of pAW-50 GS into species XL1-Blue. Transformed species spread onto ampicillin plates
-
-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.
+
 +
-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.
-
2013/03/28
+
-XL1-Blue colonies picked and screened. Purification of pAW-50 mCherry from colonies, and stored in -20°C freezer.
-
-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
+
'''June 2013'''
-
-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
+
-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock
-
-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
+
-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.
-
-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
+
-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector
-
-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
+
'''July 2013'''
-
-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.
+
 +
-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.
-
[[[[[SUMMER SEMESTER]]]]]]]]]]]]]
+
-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.

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.

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