Once we had developed a project design, designed our constructs, and sent them for synthesis we moved onto the first phase of our methods; inserting the synthesized constructs into a plasmid (for the specific protocols used, please visit the Notebook page). Following the project design phase, the designed constructs were sent for synthesis. While 6 constructs were initially designed, only 4 were able to be synthesized due to size constraints and limitations in synthesis of such long DNA sequences. Due to delays in synthesis, 3 constructs were initially delivered (labeled as Constructs 1, 2, 3). As the 4th construct took over 1 month to be synthesized and delivered, it was not possible to clone this construct into yeast (we did not have access to a microbiology lab after early August and our schools did not have the resources for yeast cloning). Below is the first phase of methods: inserting the synthesized constructs into plasmids. In the above graphic, the first part of the cloning process is summarized.
Amplification PCR
As synthesis often yields a relatively small quantity of DNA, the first step was amplification polymerase chain reaction, which uses primers (small pieces of DNA) to create more copies of the DNA. Synthesized constructs were reconstituted in TE buffer, then combined with PCR reagents (primers, DNTPs, Taq polymerase buffer, Taq polymerase enzyme), before being run in the thermocycler.
Restriction Digest
Following confirmation that the amplification PCR was successful, the PCR products were purified using a PCR purification kit. The amplified DNA constructs from the PCR, and two plasmids were digested using restriction enzymes. Restriction digest ‘cuts’ the DNA with enzymes, which results in ‘sticky ends’ on the constructs and corresponding plasmid. These corresponding ends enable the construct to be inserted into the plasmid. The two yeast plasmids used were pRS426 and pAG36. The pSB1C3 plasmid was also cut with restriction enzymes, so that the constructs could be inserted for submission to the Parts Registry. For what enzymes were used for each construct/plasmid, and exact quantities of reagents, please see the Notebook. All three constructs and 3 plasmids were digested correctly, verified with a gel.
Restriction Digest Purification
After confirmation that the restriction digest was successful, the DNA samples were purified. The constructs were PCR purified and the plasmids were gel extracted. The DNA was purified to remove the unwanted pieces of DNA leftover from the restriction digest. These unwanted pieces of DNA could prevent the construct from being properly inserted into the plasmid. The two methods of purification were used because of the different sizes of the pieces of DNA that were left after the restriction digest. The digested constructs had only very small pieces of DNA cut by the enzymes, these small pieces would be washed away during purification. The digested plasmids had large pieces of DNA several thousand base pairs long, thus these pieces had to first be separated by size with gel electrophoresis, then the desired piece of DNA was cut out and purified. This process prevents the plasmid from ligating back on itself, without the construct inside, during ligation.
Ligation
The purified constructs and plasmids were combined with a ligase enzyme and buffer. Constructs 1 and 2, which do not have a promoter, were ligated into the pAG36 yeast plasmid, which contains a TEF1 constitutive promoter. Construct 3, which contains a promoter, was ligated into the pRS426 yeast plasmid. All three constructs were ligated into the pSB1C3 plasmid.In the above graphic, the second part of the cloning process is summarized.
E.coli Transformation
After ligation, the plasmids containing our construct were transformed into E.coli. The yeast plasmids had to be transformed to E.coli first, in order to verify that the plasmid did indeed contain the construct. Competent E.coli cells were combined with the DNA samples, heat shocked, then plated onto agar plates with appropriate antibiotics. The antibiotics serve as a selectable marker for the E.coli cells that took up the plasmid. After letting the plates incubate for 24 hours, colonies were present on all plates.
Colony PCR
While the use of antibiotic resistance as a selectable marker eliminates E.coli cells that did not take up the plasmid, colony PCR must be used to determine if the plasmid inside of the cells actually contains the construct. Colony PCR uses primers that bind to the plasmid, just above and below where the construct is supposed to be. During the PCR reaction, many copies of the section of DNA between the two primers is made, and gel electrophoresis verifies how long the section of DNA is. If the plasmid does not contain the construct, then this piece of DNA will be very short. However if it does contain the plasmid, then the section of DNA will be about the same length as the construct. This method is used to ensure that only plasmids with the construct are transformed to yeast and submitted to the Registry. The PCR reagents used were: forward and reverse primers, DNTPs, Taq polymerase enzyme, Taq buffer, and water. Colonies chosen with toothpicks were stirred into the mix of reagents, and then used to inoculate liquid cultures of LB broth. These liquid cultures were set aside in the incubator, to be used the next day for miniprepping the cells. To ensure that at least one colony from each plate was successful, 7-8 colonies per plate were selected for colony PCR. After running the samples through the thermocycler, and gel was run on the samples. The gel confirmed that the constructs 1 and 2 had been properly inserted into the pAG36 vector, while construct 3 had not.
Miniprep
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Yeast Transformation
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Testing
Phase 1: Testing in Known Starch Concentrations
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Short Term Testing
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Long Term Testing
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Phase 2: Testing in Industrial Water Samples
Prototype
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Testing of Samples
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Phase 3: Cell Growth Testing
Testing in YPD Media
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Testing in Starch Media
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