Connected Purdue team to a professor at MSU for use of a GC/Mass Spectometry instrument

Quantification of Benzene via GC/Mass Spectrometry

Dates of Collaboration: 10/7/17-10/16/17

Where: GC/Mass Spec Facility- MSU

-Dr. Dan Jones

Preparing Phage Lysate

Purpose- The overall genetic construct used to degrade benzene into acetyl-coA and pyruvate consists of 9 enzymes, and was broken into two plasmids to increase the success rate of cloning. Purdue only has been able to clone the first of these two plasmids into E. coli, Upper Operon (UO). The upper operon expresses several enzymes pertaining to benzene degradation, however it does not express the protein transporter necessary for cellular benzene uptake. To get around this obstacle, you will test the induced cellular lysate instead of the living culture via GC. At the end of this protocol, you should have 30mL of uninduced cellular lysate and 30mL of induced cellular lysate.

Materials
• aTc (inducing agent)
• Tris-HCl
• NADH
• 2 autoclaved 125ml Erlenmeyer flasks
• LB broth
• Chloramphenicol

Equipment
• Incubator with shaker
• Spectrophotometer (with cuvettes if necessary)
• Sonicator

Protocol

Day 1
1. Inoculate a culture of UO E. coli in LB with chloramphenicol, CM, (25mg/L) from the provided stock culture as well as a culture of wild type E. coli and incubate at 37°C while shaking at approximately 250rpm overnight
(25mg)(1L/1000mL)(1mL/30mg)(10mL)= 0.0083333mL=> 8.33uL of MSU’s 30mg CM
10mL LB + 50uL culture+ 8.33uL CM
*CM is to only be added to UO, NOT the wild type
Day 2
1. In an autoclaved 125ml Erlenmeyer flask, aseptically dispense the following:
• 35mL LB broth
• Chloramphenicol to achieve 25mg/L
• .75mL UO culture from the previous day
In another autoclaved 125ml Erlenmeyer flask, aseptically dispense the following:
• 35mL LB
• 1.75ml wild type E. coli culture from the previous day
• CM (ONLY FOR UO-- to acheive 25mg/L)
For the Wild Type E. coli (WT) only: Incubate overnight at <35°C while shaking at approximately 250rpm
For the UO only: Incubate at 37°C while shaking at approximately 250rpm for 1.5-2.5 hours. After 1.5 hours, check the OD600 periodically until an absorbance of 0.4-0.6 is obtained
Once the desired UO OD600 is achieved, add 1ul of aTc per ml of culture Incubate the UO at 37°C while shaking at approximately 250rpm overnight

Notes: When growing to OD for UO…. After the first 1.5 hours: OD was 0.529

1. Measure the OD600 of both cultures (you will need to dilute a small volume from each sample, as absorbance does not follow a linear regression when OD600 > 1) Note: The cultures were around an OD of .980
2. Aliquot out the equivalent of 10mL of cells (of both cultures) of OD600 = 1 into individual 15mL conical tubes (in triplicate, 6 tubes total)
3. Centrifuge at top speed for 5 minutes
4. After centrifugation, cells were resuspended in 9mL 1M Tris-HCl (pH 7.5) and then transferred to 50mL centrifuge tubes
5. Of each culture (UO and WT) there was one 50mL centrifuge tube and 50mL of Tris was added in total to the cultures/resuspended to ensure more vials for sampling Note: The reason this was done was to ensure that benzene would not evaporate from multiple punctures in the caps; therefore, there were multiple samples to test of each culture/plasmid.
Lyse cells via sonication Note: This was not done due to concerns from Dr. Jones regarding the activity of enzymes and whether or not they would metabolize Benzene after sonification. Also, there was not much notice on the use of Sonification in order to sign up ahead of time to use the machine and to make sure to have access to the building.
6. Add 1mL 50 mM NADH per tube (will be provided) 500uL added to each sample/tube (refer to the Quantification of Benzene protocol below)

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Purpose As a metric in determining the effectiveness of the modified E. Coli to degrade Benzene, it is necessary to quantify and measure the rate at which benzene disappears from a closed system. Materials ● (6) 20 mL headspace vials ● LB Broth ● 99.8% Benzene (2mL) Equipment ● 0.5uL-10uL Pipette [B025] ● 2uL-20uL Pipette [B025] ● 100uL-1000uL Pipette [B025] ● Pipette-boy [B025] ● Incubator [B025] ● Gas Chromatograph Protocol Phase I: GC Optimization. Prepare Standard Solutions For each standard, mix in a 20mL headspace vial and seal The samples will be tested at 125ug/mL of benzene, so the following standards should be prepared by adding benzene to 10mL of Tris/HCl pH 7.5 broth: Blank 20ug/mL – 0.23uL pure benzene 50ug/mL – 2.29uL pure benzene 80ug/mL – 3.66uL pure benzene 110ug/mL – 5.04uL pure benzene 140ug/mL – 6.41uL pure benzene 2. Create Standard Curve Phase II: Cell Lysate Degradation Quantification Prepare Cultures for Headspace Extraction In a 20 mL headspace vial add: 5.0 mL of culture (WT and UO) 2.86uL of pure benzene (WT and UO) 500uL NADH This was done 9 times for each culture (total of 18 tubes) With the septum in place, tighten the screw cap 2. Gas Chromatography Using an autosampler, determine the amount of benzene in each headspace vial every 2-6 hours for 1-2 days