Team:British Columbia/DSZNotebook

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Latest revision as of 03:48, 4 October 2012

British Columbia - 2012.igem.org

Sept 8 & Sept 9

To obtain a control for our project which distributes the DszABC operon amongst three different auxotrophs, we needed to show the rate of desulfurization (removal of dibenzothiophene) when the entire DszABC pathway is transformed into a single host. To perform this experiment, we grew a culture of E.coli containing the optimized plasmid for the DszABC pathway cloned from Rhodococcus erythropolis IGTS8. This construct was obtained from Dr.Keasling and was the source for our DszC biobrick.

An overnight culture for this E.coli cloned with the DszABC operon was grown overnight in 5mL culture of LB. We inoculated the overnight culture in 200mL culture of LB in a 500mL flask so that we have a starting O.D600 is 0.05. Prior to inoculation, dibenzothiophene (DBT) was dissolved in DMSO at a concentration 100ppm. This was added to the 200mL culture such that it contains 2% DMSO. For a control, we inoculated an E.coli K12 strain without the plasmid, which should not contain the necessary enzyme of biodesulfurization.

Next, we incubated both cultures at 37°C shaking at 200 rpm. To obtain a representation of DBT removal over time, we extracted 25mL of each culture at O.D 600 of 0.3, 0.7, and 1.0 intervals. Once obtained, we prepared our sample for HPLC analysis.

Supernatant was extracted by spinning down cells at 1600g for 10min. The supernatant was then acidified to pH of 2.0 with 6N HCl for efficient extraction of solutes. We then extracted the supernatant with equal volume of ethyl acetate. The extract was then dried with nitrogen gas and re-suspended in mobile phase with 80% acetonitrile. The resuspended extract was lastly filtered using a 0.45 μm PTFE or nylon filter. These samples were then run on an HPLC using a C18 column (150 x 3 mm) at a flow rate of 0.8 ml/min. The samples were monitored at 280 nm with identification of DBT and its retention time.

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-'''June 15'''
+'''Sept 8 & Sept 9'''
-In order to test the desulfurization ability of our E coli consortia, we may have to grow them in some organic solvent. There is a part on the registry which is supposed to convey solvent tolerance, so we attempted to do a solvent tolerance assay using pentane and gasoline. The cells that we wanted to grow showed no growth for the first 5 hours. We decided to forgo the experiment until later on.
+To obtain a control for our project which distributes the DszABC operon amongst three different auxotrophs, we needed to show the rate of desulfurization (removal of dibenzothiophene) when the entire DszABC pathway is transformed into a single host. To perform this experiment, we grew a culture of E.coli containing the optimized plasmid for the DszABC pathway cloned from Rhodococcus erythropolis IGTS8. This construct was obtained from Dr.Keasling and was the source for our DszC biobrick.
-- [[User:jacobtoth|Jacob Toth]]
-As for the organic solvent tolerance assay, indeed there has been no growing sign in the transformed culture (with 3I plasmid) for the first few hours, and it was left in the 37 degree room over night.
+An overnight culture for this E.coli cloned with the DszABC operon was grown overnight in 5mL culture of LB. We inoculated the overnight culture in 200mL culture of LB in a 500mL flask so that we have a starting O.D600 is 0.05. Prior to inoculation, dibenzothiophene (DBT) was dissolved in DMSO at a concentration 100ppm. This was added to the 200mL culture such that it contains 2% DMSO. For a control, we inoculated an E.coli K12 strain without the plasmid, which should not contain the necessary enzyme of biodesulfurization.
-and for the controlled cultures, they were growing as expected when the organic solvents (Pentane and Octane) were not introduced, the experiment was continued (with the disruption of Jacob mentioned before). The organic solvent were added into controlled cultures at different concentrations (0%, 5%, 10%, and 15%), and their growth were monitored roughly each hour for the first few hours, and when the disruption occurred the cultures were taken out of the 37 degree room and stored in the 4 degree room at 5 p.m.
+Next, we incubated both cultures at 37°C shaking at 200 rpm. To obtain a representation of DBT removal over time, we extracted 25mL of each culture at O.D 600 of 0.3, 0.7, and 1.0 intervals. Once obtained, we prepared our sample for HPLC analysis.
-I returned to the Lab at 8:30 p.m., and each culture were examined for OD readings again, and the cultures with Pentane and Octane were incubated in the incubator with vacuums at 37 degree for 1 hour and 30 minutes respectively. Then the OD were read again at 9:30 p.m..
+Supernatant was extracted by spinning down cells at 1600g for 10min. The supernatant was then acidified to pH of 2.0 with 6N HCl for efficient extraction of solutes. We then extracted the supernatant with equal volume of ethyl acetate. The extract was then dried with nitrogen gas and re-suspended in mobile phase with 80% acetonitrile. The resuspended extract was lastly filtered using a 0.45 μm PTFE or nylon filter. These samples were then run on an HPLC using a C18 column (150 x 3 mm) at a flow rate of 0.8 ml/min. The samples were monitored at 280 nm with identification of DBT and its retention time.
-[[File:British_Columbia_Octane Tolerance_Control.png|900x1000px]]
-*Figure 2. Octane Tolerance Control Test
-[[File:British_Columbia_Pentane Tolerance_Control.png|900x1000px]]
-*Figure 3. Pentane Tolerance Control Test
--  [[User:Ruichen|Ruichen]]
-'''June 16'''
-The cultures with 3I (solvent tolerance) plasmid that left for growth in the 37 degree room actually did turn a bit cloudy (6:00 p.m.).
-Their ODs were then measured with a blank LB for reference, and the detailed info will be updated.
-For the remaining cultures, 100µl of each culture was tested on Chlor plates with an control of K12 (20µl)  to see if they are actually the cell cultures that of our interest, and also testing the chlo plates that are newly made, and the remaining cultures were stored in the 4 degree room for potential future usage.
-The plates were left in the 37 degree room, and will be collected by Joe and stored in the 4 degree room.
-- [[User:Ruichen|Ruichen]]
-'''June 18'''
-We made TB broth in order to grow our Rhodococcus strain, which harbors the desulfurization plasmid.
-- [[User:jacobtoth|Jacob Toth]]
-'''June 20'''
-Plated the cultures with 3I plasmid on Kan for control and Chlor for testing purpose.
-- [[User:Ruichen|Ruichen]]
-After consultation, we moved the IGTS8, IGTS9, and Rhodococcus JHV1 strains that were growing on terrific broth and also ones growing on LB from the 37C shaker to one at 30C.
-- [[User:Tingchiawong|Tingchiawong]]
-'''June 21'''
-The culture has been proven to be transformed successfully with the 3I plasmid! They grew colonies on the Chlor plate and not the Kan plate.
-Learned how to miniprep
-Checked out some cool posters for the microbiology Conference, and found that there is a assay of interest. The assay (2,6 DCPIP) helps organisms to grow on a diesel contaminated soil.
-- [[User:Ruichen|Ruichen]]
-'''June 22'''
-Wanted to design mutagenesis primers for our Dsz genes, but couldn't remember what software to use. For future reference:
-NCBI for FASTA
-Nebcutter to find sites
-Virtual Ribosome for associated peptide
-Wikipedia for codon table
-PrimerX for basic design.
-Joe appears to be successfully growing IGTS8 (Rhodococcus) and IGTS9 (Bacillus) in TB at 30°C, and Pseudomonas putida at 37°. He also reports being unsuccessful in doing a cPCR for the dszD gene.
-- [[User:jacobtoth|Jacob Toth]]
-The IGTS8 and IGTS9 cell cultures in both terrific broth and LB grew dense and cloudy at 30C.  Glycerol stocks will be made out of these.
-'''June 23'''
-Made competent ''Pseudomonas putida'' cells using the [[Competent Cell Production]] protocol.
-'''June 25'''
-Designed primers for SDM of dszB gene
-Had a Skype meeting with Calgary iGEM team
--[[User:Ruichen|Ruichen]]
-'''June 26'''
-Designed primers for SDM of dszC gene.
-- [[User:jacobtoth|Jacob Toth]]
-'''July 15'''
-We ordered the primers for DszC mutagenesis.
-- [[User:jacobtoth|Jacob Toth]]
-'''August 12'''
-We received the DszA forward primer, and ran a successful PCR. We then did a restriction digest on the product. It will be ligated as well in the near future.
-- [[User:jacobtoth|Jacob Toth]]
-'''August 16'''
-We ligated the DszA with pSB1C3 and transformed. A cPCR on the resulting colonies gave no result. This may have been due to an unoptimized Taq protocol,and might be worth repeating.
-- [[User:jacobtoth|Jacob Toth]]
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-- [[User:Ruichen|Ruichen]]