Team:Nevada/Notebook/Weeks58

Calender Weeks 5-8
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Week 5 - June 27th- July 3rd

E. Coli

Pyruvate Decarboxylase & Alcohol Dehydrogenase

JC: To insert PDC/ADH genes into the σ70 constitutive promoter plasmid, 0.5ug of σ70/pSB1A3 (2190bp) was digested with SpeI and PstI and 0.5ug of PDC/ADH/pSB1C3 (5124bp) was digested with XbaI and PstI. These digestions cut out the red fluorescent protein (rfp) tag on pSB1A3 (rfp=885bp) to aid in colony selection. Digestions were run on a 1% agarose gel, and bands obtained confirmed PDC/ADH (3054bp), pSB1C3 (2070bp), σ70/pSB1A3 (2190), and rfp (885bp). Incomplete digestion of PDC/ADH/pSB1C3 was apparent for sample #8, so the sample was re-digested before ligation.

PDC/ADH-XbaI/PstI and σ70/pSB1A3-SpeI/PstI were ligated and transformed into NEB10 β cells. Single colonies from LB-Ampicillin plates were selected and tested on LB-Amp and LB-Chlor plates. No usable colonies were obtained (all were light pink and grew on both LB-Chl and LB-Amp plates).

Cyano
Procedure: DH5α cells were transformed with the promoters K390015 (petBD+RBS), K390016, and K390017; the integration vector K654056; GLF; and INV. All genes were contained in the plasmid pSB1C3. Cells were selected for on chloramphenicol plates and cultured in LBccm broth. Plasmid DNA was isolated from the transformants using a QIAgen miniprep kit. Isolated plasmid was digested with EcoRI and PstI to ensure that the correct plasmid transformations took place.

Results/Discussion: The EcoRI and PstI digests did not confirm successful plasmid transformations. This seems to be due to a defect in the restriction enzymes. In order to advance in our research, these isolated plasmids will be submitted for sequencing.

Enzymology
This week we ran the ethanol test assay using ADH. Results were negative as ethanol standards of low concentrations (<20µM) were not being detected. Only when high concentrations of ethanol were added was there any absorbance correlating to ADH activity. We therefore concluded that the ADH enzyme was not sensitive enough to detect ethanol within the concentration ranges we were expecting, and opt to use a more sensitive Alcohol Oxidase enzyme instead. The alternative enzyme was ordered and will be stored until testing. A rough draft protocol for the alcohol oxidase assay was also written up and buffers were prepped.

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Week 6 - July 4th-10th

E. Coli

Pyruvate Decarboxylase & Alcohol Dehydrogenase

PDC/ADH was re-digested with XbaI/PstI and run on a 1% agarose gel to confirm digestion. Bands obtained confirmed PDC/ADH (3054bp), and pSB1C3 (2070bp). PDC/ADH-XbaI/PstI and σ70/pSB1A3-SpeI/PstI were re-ligated and transformed into NEB10 β cells.

Cyano
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Enzymology
Week 6-7 This week we ordered a Free Fatty Acid Assay Kit from BioSystems as we anticipate that our E. coli modified to produce and secrete free fatty acids part is complete. While I waited for my kit to arrive I helped around the lab and participated in a western blot. The western blot was to detect a Histidine tag on the Bay Laurel thioesterase gene. The blot was attempted and failed three times before we concluded we were using a defective transfer buffer.

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Week 7 - July 11th-17th

E. Coli

Pyruvate Decarboxylase & Alcohol Dehydrogenase

Single colonies from LB-Amp plates were selected and tested on LB-Amp and LB-Chlor plates. Liquid cultures grown in LB-Amp and minipreps and nanodrop analysis performed.

To verify presence of PDC/ADH genes with the σ70 constitutive promoter, 0.5ug of PDC/ADH/σ70/pSB1A3 was digested with EcoRI and PstI. Digests were run on a 1% agarose gel, and bands confirmed σ70/PDC/ADH (3089bp) and pSB1A3 (2155bp).

Cyano
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Enzymology
See week 6

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Week 8 - July 18th-24th

E. Coli
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Cyano
7/17/11 – PCR temp gradient was created for ThiE solution, used New England Biolabs phusion high-fidelity DNA polymerase protocol. Temp gradient ranges from 72-50 degrees Celsius. Controls of only DNA and only Primer were run with the samples. 10 ul samples were run for each temp gradient
7/18/11 - PCR results were run through a 1% agarose gel. Best results came from ‘F’ which ran at 54.3 Celsius. The DNA from this band was extracted and purified using QIAGEN Gel Extraction Kit
7/19/11 – PCR 50 ul of ThiE at 54.3 Celsius for backup stock. TOPO cloned ThiE that was extracted from gel yesterday. Plated TOPO cells at 100 ul, 75 ul, 50 ul and 25 ul concentrations then placed in incubator overnight
PCR reactions only need one control, of just the primers without DNA.
7/20/11 – 5 colonies of TOPO bacteria grew overnight. Cultured all 5 for purification and confirmation of proper insert (ThiE) Ran gel to confirm that 50 ul ThiE PCR worked, nice clean band at 1 kb marker, saved the rest of the 50 ul stock in -20 C freezer for further use. PCR done on the additional pieces (CmR, PetBD and GLF) used same temp gradient as done with ThiE. Will run a gel of these tomorrow to see what optimal temp is for each sample. If clean bands are seen, we will perform a gel purification and re-PCR those samples. Spoke with Shintani, these additional pieces should just be amplified through PCR until a clean stock is created, then this stock will be used in Gibson.
7/21/11 – Miniprep purified TOPO cultures. Sample “1” did not bloom in culture but grew on stock plate. Purified “2-5” and will save “1” as a backup if no other colonies have the correct insert. Ran temp gradient samples through gels.
GLF had very light bands at 67.6 and 63.5 Celsius temps, the average temp of those two is 65.5 – will try to run more DNA at this temp to see if we can get stronger bands. Maybe increase DNA concentration in solution.
*Shintani gave me his master stock solution of GLF (in puc vector) for next run of PCR. Diluted 1:9 of his solution and stored in freezer in PCR tube (with orange label) will try to PCR this stock at 65.5.
PetBD worked really well at several temps but 63.5 Celsius appears to be the best temp, strong band that was very clean. This worked out so well I might try just running a 50 ul solution at this temp and saving it as backup stock. Confirm the same clean band with a 5 ul run of solution through gel.
CmR did not work at all  I was told K56 was the integration vector sent from Utah State but was unable to confirm that in any of my team’s notebooks. Will double check with David today and see if he has that same number in his note book. If it is correct, maybe need to play with DNA concentration and temps again.
*E. coli group has extra CmR, will ask Megan to bring over some of this stock (pSB1C3 – iGEM vector) for next PCR. Shintani thinks that maybe vials were switched in the beginning (KmR PCR also not working) or something went wrong during purification.
With better stock will run temp gradient PCR on CmR again.
7/22/11: Gel for GLF at 65.5C yielded no bands. Ran PCR of GLF at 63.5C. Faint bands, attempted gel purification, will run confirmation gel on Monday. Ran PCR for PETBD (50ul), which also yielded no bands. Will run another gel for a shorter time period on Monday. Will discuss options/next steps on Monday with the group.

Enzymology
This week we received the FFA assay kit and tested two BTE/PDC transformed cultures (+ 4 negative sigma70 controls); pellets and supernatants both were tested. Results were positive for one of the cultures and results suggested that fatty acids were present in the supernatant as opposed to the cell itself. We decided to do a time course growth on the identified culture.

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