Team:Minnesota/Experiments
Minnesota
Experiment 1: Creation of the FitD Plasmid.
The E. coli will be transformed with the PBAD18 plasmid and grown in the absence of arabinose to ensure that transcription of the plasmid genes, including cytolysin FitD cannot occur, thus limiting the chance of prematurely killing E. coli cells in the event that cytolysin FitD is toxic to the cells. Following transformation, the PBAD18 E. coli will be grown in the presence of arabinose, thus allowing transcription of the genes on the PBAD18 plasmid, including FitD. If the E. coli cells survive growth in arabinose, a coomassie gel will be run and the amount of cytolysin FitD will be quantified.
Experiment 2: Creation of Purine Auxotrophy System
Design and synthesize the PurA plasmid. Transform E. coli with plasmid. To test the delayed auxotrophy system, the UMN iGEM team will culture the engineered E. coli in a series of culture tubes without IPTG. At various time points, the turbidity of the culture will be measured using spectroscopy in order to quantify the number of viable cells. Cultures will also be plated out and CFU/ml will be determined. If the delayed auxotrophy system works, it is expected that the number of viable cells will decrease in relation to time.
Experiment 3: Co-Transform Purine and FitD Plasmid
The two plasmids will be co-transformed into E. coli. The E. coli will then be streaked on media containing kanamycin and ampicillin in order to select for the cells that contain both plasmids. To test the efficiency of the co-transfection, an additional plasmid will be designed that has both of the expression systems on it. These two systems will be tested to see which is most efficient and which has the best control for release in the environment.
Experiment 4: LD50 of FitD system
The Minnesota Aquatic Invasive Species Research Center (MAISRC), which works in partnership with the University of Minnesota, has agreed to provide us with zebra mussels so an experiment to determine LD50 can be run on the engineered E. coli.
