Team:UGA-Georgia/Notebook

From 2013.igem.org

(Difference between revisions)
(→Methanococcus Lab)
(→E. coli Lab)
 
(16 intermediate revisions not shown)
Line 7: Line 7:
Instructor: ZHE LYU
Instructor: ZHE LYU
-
'''Tasks completed before 2013/02/20'''  
+
 
 +
'''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.
+
-
+
-
-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
+
-Purification of GS, AT and GS+AT via revival of frozen stocks and plating of sub-cultures.
-
Appendix of protocols, I Preparation of Complex Medium for Methanococci
+
-
(McC).  
+
-
'''2013/2/20'''
 
-
-Further preparation of media tubes via inoculation of Puromycin and
+
'''April 2013'''
-
pressurization to 40 psi using H2/CO2 gas. Stored at -20C.
+
-
-Preparation of samples for SDS gel.
+
-Further continuation of purification of GS, AT and GS+AT via picking colonies, creating sub-cultures and plating.
-
+
-
-Vector (control) samples made at 1x dilution and 5x dilution
+
-
-Geranyl Synthase (GS) plasmid samples made at 1x and 5x dilution
+
-Test of Puromycin strength.
-
-An SDS gel was run using both dilutions of vector and GS samples, along with a
+
-Creation of frozen stocks of purified GS, AT and GS+AT transformants.
-
protein marker.
+
-
'''2013/2/26'''
 
-
-Four media tubes were treated with 0.1 mL Na2S solution each. Two of the tubes
+
'''June 2013'''
-
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
+
-Extraction of Geraniol from extra-cellular and intra-cellular content of samples and preparation for GC/MS evaluation
-
decided to perform a second gel.
+
-
'''2013/2/27'''
+
-Sequencing of pAW50-mCherry vector
-
-Reflecting upon first SDS gel, new sample dilutions were created in aim of  
+
-Analysis of pAW50-mCherry sequence
-
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
+
-Transformation of pAW50-mCherry vector into Methanococcus
-
of GS protein. Next verification of this will be to run an mRNA extract for
+
-
conclusive evidence of GS gene being transcribed 
+
 +
-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'''
-
[[[[[[[[[[[[[[[[[[[[Fig 1. ]]]]]]]]]]]]]]]]]]]
+
-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'''
-
'''2013/03/05'''
+
-Revival and PCR of all GS and AT frozen stocks to confirm insert.
-
-6 tubes of McC media created, added Na2S (0.1mL 2.5%) and Puromycin (0.5mL of  
+
--Extraction of Geraniol from extra-cellular and intra-cellular content of all GS frozen stocks and preparation for GC/MS evaluation
-
10x)
+
-
-Inoculated vector strain Methanococcus into two tubes and GS + AT into other two
 
-
tubes. 
 
 +
'''September 2013'''
-
'''2013/03/06'''
+
-Transformation of V4 into Methanococcus
-
-Solid McC media was autoclaved so gel would melt, so we could add Na2S and
+
-Testing Fluorescence of V4
-
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
+
-Purification of V4 cultures via plating of transformants, and creating sub-cultures of colonies picked
-
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'''
+
-Creation of frozen stocks of V4 in Methanococcus
-
-4 media tubes were prepared with 0.1 mL of 2.5% Na2S solution. These tubes
+
-GC/MS Evaluation and analysis (see results tab)
-
were then pressurized to 40 psi using H2/CO2 gas.
+
-
-Puromycin solution was prepared: 7.5mg of 200x Puromycin was added to 30
+
== '''E. coli Lab''' ==
-
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
+
Instructor: Rachit Jain
-
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'''  
+
'''February 2013'''
-
-200 mL liquid media was created, dispensed into 40 tubes, stoppered and capped,
+
-Training on PCR and heat shock transformation
-
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
+
'''March 2013'''
-
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
+
-PCR of GS gene
-
incubation room.
+
-
'''2013/03/26'''
+
-Cloning of GS gene into pAW-50.
-
+
-
-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
+
-Transformation of pAW-50 GS into species XL1-Blue. Transformed species spread onto ampicillin plates
-
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
-
'''2013/03/28'''
 
-
-Re-pressurize H2/CO2 cultures tubes to 40 psi.
+
'''April 2013'''
-
-Measure OD of cultures tubes hoping much better growth since 2013/03/27. The OD
+
-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.
-
measurements provided results satisfactory for plating.  
+
-
-Various appropriate dilutions of AT and GS+AT cultures were created relative to OD
+
-PCR and gel verification of mCherry gene
-
readings as described in the process from 2013/03/06.
+
-
-These diluted samples were inoculated into 6 solid media bottles for plating and plates
+
-Cloning of mCherry gene into pAW-50 vector
-
were placed into 37C incubation room.
+
-
-3 autoclaved glycerol stock bottles made 2013/03/27 were used to create frozen culture
+
-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.
-
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'''
+
-XL1-Blue colonies picked and screened. Purification of pAW-50 mCherry from colonies, and stored in -20°C freezer.
-
+
-
-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
+
'''June 2013'''
-
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
+
-Transformation of pAW-50 mCherry into XL1-Blue and creation of stock
-
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.  
+
-PCR of two mCherry sequences: one without RBS sequence (V2) and one containing RBS sequence (V4). Gel Verification of tools 1 and 2.
-
-Colonies were picked from an AT and AT+GS plate. Four colonies were taken from
+
-Digestion and cloning of V2 and V4 into pAW-50 mCherry vector
-
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'''  
+
'''July 2013'''
-
-1 mL of two different GS cultures (labeled GSF-1 and GSF-2) were inoculated in a 1 mL
+
-Purification of digested vector, ligation, and transformation of vector into XL1-Blue. Transformants plated onto ampicillin plates.
-
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
+
-V2 and V4 colonies extracted and screened. Permanent stocks of V2B and V4B stored in -80°C freezer.
-
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
+
-
 
+
-
'''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.
+
-
 
+
-
== '''E. coli Lab''' ==
+
-
 
+
-
Instructor: Rachit Jain
+

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.

Retrieved from "http://2013.igem.org/Team:UGA-Georgia/Notebook"