Team:UChicago/Experiments
Experiments
Cloning
Goals: To successfully produce the plasmids outlined in our design goals. Workflow: Quick-change PCR was used to repair the Fatty acid photodecarboxylase gene which had a single base pair deletion when the gene was received from IDT. This gene was then inserted into a Psb1C3 backbone using gibson assembly. PCR was used to amplify the presence of individual enzyme genes for FabF, FabD, FabZ and FabG from the FASyn Operon which were each individually inserted into pSB1C3 backbones using gibson assembly. Quick-Change PCR was used to edit the SigA promoter to be active in E. Coli. PCR was used to add FLAG tags to the AlkL gene and SigA promoter, then the parts were reassembled into pSB1C3 using Gibson Assembly. Many difficulties were encountered during our attempts at gibson assembly due to homology between the biobrick prefix and suffix, so a new set of primers that could replace the old set of standardized primers (VR,VF2, suffix adaptor and prefix adaptor) was created. See our results page to learn more.
Growth Curves
Goals: The growth curve data was taken to empirically measure the growth rates of Synechococcus in several conditions and implicitly determine some of the effects of the transformation of our plasmids. We took data on a control sample, a sample with the fatty acid photodecarboxylase gene inserted into a backbone with chloramphenicol resistance (Alk), a sample with the AlkL channel protein gene inserted into a backbone with ampicillin resistance (AlkL), and a sample with both plasmids transformed and selected for by both antibiotics. Protocol: 1. Transform desired plasmids into E. coli and plate them. 2. Make overnight cultures, with 3 or 4 replicates for each plasmid. 3. The following day, take the OD600 of each culture. Make an approximately 10X dilution to get all of the cultures down to a similar OD. 4. Take a starting OD. 5. Incubate the diluted cultures in a shaker at 37°C under blue light for 2 hours. 6. After 2 hours, take the OD again. 7. Continue to take the OD every 2 hours.
Gas Chromatography Mass Spectrometry
Goals: The goal for this procedure was to assess the production of alkanes in E. Coli containing the Alk Operon. Protocol: The Alk-operon was transformed into E.coli and those E.coli were grown in an LB broth overnight in a photosynthetic incubator where they were kept overnight at 37C and exposed to a white light source for the duration of the night. The overnight cultures were set for 5 days and stored at room temperature. Next the overnight cultures were centrifuged down and the pellets were resuspended in in 350 ml of STET buffer (10 mM Tris-HCl, pH 8.0, with 0.1 M NaCl, 1 mM EDTA, and 5% [w/v] TRITON“ X-100). Next, 25 ml of a freshly prepared lysozyme solution (10 mg/ml in 10 mM Tris-HCl, pH 8.0) was added to the samples. After a 30 minute incubation at 37C, the lysis mixture was centrifuged at 14,000 Xg for 10 minutes. The Gas Chromatography Mass Spectroscopy (GCMS) was performed with the solvent Hexane. 200ul of Hexane was added to lysis mixture and then 100ul of that new mixture was pipetted into a vial containing another 900ul of Hexane. This sample was compared to a negative control in which the identical procedure was carried out on transformed E. coli with a PSB1C3 empty vector. The sample was also compared to a positive control that contained 80ul of an Alkane standard ladder dissolved in 400uL. All three samples were run through Agilent 7200B Q-Tof GCMS and the results were analyzed to assess the production of alkanes in the E. coli containing the Alk Operon.
Fluorescence Assay
Goals: The goal of our fluorescence assays was to characterize the ribosome binding site with part number BBa_J61100. The specific goal was to characterize green fluorescent protein expression in E. Coli Protocol: The protocol followed was developed in iGEM's 2018 interlab study, and can be found here.
Western Blot
Goals: Western Blots were performed to ensure proper expression of the AlkL protein. AlkL was the only FLAG-tagged protein in this experiment. Protocol: