Team:Nevada/Notebook/Weeks912

Calender Weeks 9-12
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Week 9 - July 25th-31st

E. Coli

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Cyano
7/25/11: Chloramphenicol vector was digested with EcoRI and checked on 0.7% gel. The results for this gel were good. ThiE/Topo Vector was digest with EcoRI and PstI and checked on a 0.7% gel. The gel was not very clean, so we will re-run tomorrow (7/26). The PetBD and GLF PCR was done at 63.5C. Positive control/primer controls were added to the PCR reaction. After confirmation of ThiE it will be put into plasmid PSB1C3 and sequenced. 7/26/11: Gels for PCR products of PetBD and GLF were run with no results. We believe this is due to errors in the master mix. ThiE digest was re-run on a 0.7% gel. The gel showed that the ThiE was not digested. (7/27). Ran PCR reaction on ThiE gel purification 7/18/11 with positive control and primer control at 54.3C for Topo Cloning tomorrow (7/27). 7/27/11: Ran gel on + control, Primer control and ThiE to be used for Topo cloning. Gel was completely empty aside from ladders. Did digest on ThiE in Topo Vector with ecoRI. Results showed that we have an empty topo Vector on all three digests (#'s 2, 3 and 5).

Enzymology
This week we performed our time course growth on the positive culture sample taking samples every two hours with an overnight culture as well. We performed the FFA assay on every time course sample using the BioAssay Systems FFA assay kit with slight modifications to the manufactures protocol. The results were negative for all samples which could have possibly been due to improper storage of the assay kit. Another assay kit was ordered as samples were to be further tested.

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Week 10 - August 1st-7th

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 10-11 We began to focus on proof of ethanol production as the E.coli part modified to produce ADH and PDC neared completion. We purchased an Ethanol Assay Kit once again from BioAssay Systems and performed the assay on eight different cell lines plus two negative controls (sigma70 promoters) and four standards (included in the kit). Results proved to be negative as there was no visible color change in the samples and there absorbencies were no higher than that of the negative control. We opt to take a step back and determine where the problem was. The sequencing report determined that both the ADH and the PDC genes were present, but were they being expressed? We chose to use aldehyde detecting plates to determine if the PDC enzyme was begin produced. We also decided to write up a protocol to lyse the modified cells themselves and assay for ADH activity instead of ethanol presence.

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Week 11 - August 8th-14th

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
This week we ran the ADH activity assay testing five different ADH/PDC cell lines and one positive (ADH @ 0.75u/ml) and negative (sigma70) standard. We used a standard lysis buffer to lyse the cells and lysate was used as the enzyme additive for the assay. Unfortunately, there was still no sign of ADH activity. Although the positive control tested positive in both assay and lysis buffer, we decided to view sources with similar experiments to get an idea of what conditions we should be running the cell lysis and assay at. Also prepped was an alcohol oxidase assay which will be used to detect ethanol (if we get that far)!

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Week 12 - August 15th-21st

E. Coli
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Cyano
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Enzymology
This week was our third FFA assay using the new FA assay kit from BioAssay systems testing fresh samples from different cell lines than from before. Three of the five cultures tested positive for fatty acids. Fresh overnight cultures of these cell lines will be re-assayed along with two sigma70 negative controls. We performed our final FFA assay (using the assay kit) on 4 samples that had previously tested positive plus three new samples and two negative (sigma70) controls. All seven samples tested positive with significantly higher absorbencies (α to concentration) than background and the negative controls. Those samples, plus two fresh negative controls were prepped for GC analysis (extracted with hexane), and GD analysis is scheduled for next week.

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