Team:NorthernBC/Experiments
We had our genes sequenced by IDT, the editing beforehand included removing illegal restriction sites within the gene, as well as adding the iGEM prefix and suffix. In addition to these required steps, we optimized the genes for E. coli and added a multiple cloning site (MCS) at the end of the YiaT protein, so that any protein could be fused, making YiaT a multi-functional platform.
We used the 3A ligation protocol to create our plasmids, we chose a constitutive promoter because of the nature of our project, we needed the protein to be expressed consistently to meet our goals, we chose to use the double terminator, as it has been proven many times to work effectively. The order of genes in the plasmid is Promoter and ribosome binding site, YiaT protein with MCS, and then fusion protein. The first fusion protein we used was a green fluorescent protein terminator, which we used to detect localization of the protein fusion, to ensure that expression was occurring as intended and the green fluorescent protein was external to the cell. Our other fusion proteins were two copper binding proteins, Cup1 and copper metallothionein.
We used Qiagen QIAprep spin miniprep columns to purify plasmids. We used colony PCR, ethidium bromide agarose gels and ultra violet illuminators to visualize endonuclease digests and ligations.
Fluorescent protein localization
In order to analyze membrane localization of the cID1021 (YiaT-MCS fusion protein), a green fluorescent protein was attached to the fusion protein and analyzed by microscopy. The parts used are described in Table 1. First cID1021, cID1025 (GFP-termintor), and cID1017 (promoter-RBS) were transformed into DH5α competent cells, grown in an over night broth containing chloramphenicol and LB (BactoTM Agar) and purified using a Qiagen Miniprep kit.
The purified DNA was digested and ligated using the Biobrick Assembly Protocol from New England Biolabs. First, the upstream part, cID1017, was digested with EcoRI and SpeI. The downstream part, cID1021, was digested using Xbal and PstI. Lastly, the destination plasmid, cID1013 (pSB1K3), was digested using EcoRI and PstI. All enzymes came from New England Biolabs. The parts were left at 37oC for 60min and 80oC for 20min.
The digested products were run on a 1% agarose gel to confirm that the digestion had been successful. The parts were ligated using a 1:3 vector to insert molar ratio. 50ng of the vector was used. The ligation was transformed and plated on kanamycin-LB plates. The ligation was purified using the Qiagen Miniprep kit and confirmed by gel electrophoresis. The process was repeated in order to ligate cID1025 and the previously ligated part into cID1022. The previously purified ligation was treated as the upstream part and was cut with EcoRI and SpeI. CID1025 was treated as the downstream part and was cut with Xball and PstI. CID1022 was the destination plasmid psB1C3 and was cut with EcoRI and PstI. CID1022 contains chloramphenicol resistance, and therefore the second round of ligations was plated on LB-chloramphenicol plates, grown up, and purified. To determine the localisation of YiaT-MCS, the purified DNA would be transformed into DH5α competent cells and analysed using an Olympus epifluorescence microscope.
Copper binding test
Copper (II) ions are blue in colour, and so the detection of them by spectroscopy is a simple procedure. Our methods to detect the binding of copper ions by our cells would be to make a standard curve of known copper concentrations, and then inoculate a copper sample with YiaT- copper binding cells, and at different time points, remove a sample, spin down the cells in the centrifuge and transfer the supernatant to the cuvette to measure the residual copper in the solution. Controls would be needed to ensure that spinning the cells does not release bound copper, or otherwise influence the results.
Transformation Protocol
Competent DH5α cells were thawed on ice and 50uL of the cells were transferred to a 2mL tube. Then 5uL of purified plasmid DNA or DNA obtained from the iGEM distribution kit was added to the 2mL tube and the resulting mixture was pipetted up and down to mix. The mixture was then incubated on ice for 30 minutes and then heat shocked for 1 minute at 37°C before another 5-minute incubation on ice. Then 200uL of LB media was added to the transformation and the mixture was incubated at 37°C for 2 hours in a shaking incubator. Next 200uL of the transformed cell mixture was used to make a spread plate on LB plates imbibed with the appropriate antibiotic. The plates were then incubated at 37°C for 18 hours to obtain individual colonies.
