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).

Procedure: KnR was to be dropped out from the pUC4k vector using an EcoRI digest. KnR would then be purified from the gel in order to use as a template for the PCR of KnR for gibson assembly. .5 ug of pUC4k was digested with EcoRI and incubated at 37 degrees C for 1 hour. The digest was ran on .7% gel at 110 V for 1 hour.

Results: The EcoRI digest was unsuccessful as indicated by cut plasmid being identical to the uncut control. Thus, the EcoRI enzyme most likely needs to be reordered.

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
After ordering newly designed primers, this week focused mostly on PCR amplification of: AGP, Kan Resistance, the promoter pETBD and Invertase. The initial amplification of AGP (ADP-glucose-pyrophosphorylase)(8/1) was performed using old primers and the fragment observed was approx. 700 bp too short. Transformation of the vector pUC4k into E. coli was performed to produce a large amount of vector for the PCR of Kan Resistance. Digestion of pUC4k with EcoRI yielded the correct 1301 bp fragment indicating the presence of Kan R in the vector. 8/3 - 8/4 PCR results showed successful amplification of Kan Resistance and pETBD, 1341 and 219 bp respectively.

Created new sterile LB stock. Poured new plates with LB + Ampicillin, LB + Chloraphenicol, and LB + Kanamycin. Ran PCR of cyanobacteria genomic DNA to isolate ThiE at 54.3C. Gel for ThiE isolation showed bands at the appropriate length, just over 1kb. Purified ThiE from the gel. Nanodrop gave readings of an average of 7.45ng/ul. Transformation of cultures will be done later in the week with blue/white selection. Waiting for new primers, as soon as they come in to the lab we will begin isolation of PetBD, GLF and Chloraphenicol. Set up PCR reactions for promoter petBD and ThiE with positive control. PetBD was successful, but with the wrong primers. ThiE still showing two distinct bands, will perform gel purification to try to clear up. Received new primers for PetBD and Chloamphenicol. Ran PCR with new primers and petBD was successful and is now ready for Gibson assembly. Chloramphenicol was not successful, will run a temperature gradient on it next week. Performed gel purification on ThiE and ran on a 1% gel. Also performed PCR reactions for GLF and Chloramphenicol and ran on a 1% gel. Only band present was for GLF, but not strong or clear.
Enzymology
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
Successful PCR of Kan R and pETBD the previous week allowed us to move forward and perform larger, 100 ul volume reactions with these two genes so that we would have a large stock with which Gibson assembly could be performed as well as a back-stock from which any further PCR of the genes could be performed. 8/8 PCR of AGP from Synechocystis yielded the correct fragment at 1360 bp. Liquid cultures of E. coli transformed with pUC57 were cultured for a large supply of vector containing the synthetic Invertase (INV) gene. Both PCR attempts (8/10 - 8/11) were unsuccessful at selected annealing temperatures.

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
Due to unsuccesful isothermal PCR of INV the previous week, a temperature gradient from 54 degrees C - 65 degrees C during the first three annealing cycles was performed. AGP was successfully amplified at 54 C, 55.57 C, 60.28 C, 61.85 C, 63.43 C and 65.0 C. The highest two annealing temperatures, 63.43 C and 65.0 C yielded the most intense bands. Larger volume reactions, 100 ul, were then run to produce large stocks of INV for Gibson Assembly and any further PCR. The four genes of interest, AGP, INV, pETBD and Kan R were then PCR purified using QIAgen kits in preparation for Gibson Assembly. 8/16 marked our first attempt at Gibson Assembly. The goal of the Gibson Assembly procedure is to create a functional operon containing KanR, pETBD, and INV (in that order) flanked on the 5' side by approx. 900 bp of AGP and on the 3' side by approx. 420 bp. Natural recombination with this construct will knockout the AGP gene leading to enhanced production of sucrose. The inclusion of KanR will be used to select bacteria transformed with the Gibson construct. While performing our first attempt at Gibson, the genes of interest were added equally in volume but not equamolarly, we believe that this is the primary reason the first attempt was unsuccessful. 8/18 Gibson attempt 2, corrections were made so that each fragment was added equamolarly, however after the protocol was performed and 5 ul of the reactions were run on agarose gels alongside stock solutions of our genes of interest, KanR was approx. 800 bp too short. As a result, KanR had to be reamplified by PCR. 8/19, KanR was successfully amplified out, an intense fragment of ~1300 bp was observed on the gel in accordance with the expected KanR fragment size.
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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