Future Lab Directions
Through trials and travails, we eliminated the crimson glow of mcherry-integrated E. coli. We were able to successfully demonstrate our system’s ability to disrupt a gene, and we have also already constructed and isolated the plasmids necessary for the next two steps of our system: phage infection and conjugation.
That is the extent of the experimental successes we may present in this competition.
However, while this year’s iGEM competition may soon be over, we’re far from finished. We’ve proven that we can disrupt genes, and in the coming months we can begin experimenting with phage infection and conjugation.
To demonstrate the viability of phage infection as a means to deliver the gene elimination machinery, we constructed the plasmid φMINT to contain both the INTEGRATE gene disruption system and an F1 phage packaging origin of replication. φMINT will be transformed into DH5α E. coli alongside the M13KO7 helper plasmid. The DH5α should then secrete phages containing φMINT into its growth media, which we will then purify to isolate the phage. We will extract the DNA from these phages and sequence it to verify the presence of φMINT. Once that is confirmed, we can dose our DH5α-F’-mcherry cells with the φMINT phage, and measure the subsequent fluorescence of the cells. If the delivery was successful, we would observe a decrease in fluorescence similar to our results from the prior gene elimination experiments.
To explore our system's capacity to propagate through a bacterial population we will conduct conjugation experiments. Conjugation experiments require the addition of the F’ origin of transfer (oriT) to our plasmid. This new plasmid, coined φMINTO, will be transformed into F’ donor cells that contain all the necessary machinery to mobilize it for transfer to recipient cells. Non-selective liquid media will be inoculated with recipient F-E.coli DH5α-mcherry cells, and its fluorescence will be measured. After confirming the presence of φMINT in a chosen F’ colony, the donor will be mixed in with the recipient cells and grown. At regular intervals, we will analyze the fluorescence of the liquid culture. If the DH5α-mcherry receives φMINTO, we should see a notable decrease in the fluorescence of the culture.
These mixtures will be done with a variety of donor to recipient ratios to discover which ratio produces the most
When we have successfully delivered φMINT/φMINTO via phage infection and conjugation individually, we can finally put the two steps together. F’ cells will be dosed with phage, then placed in media with a recipient strain containing mcherry.
As in all the experiments, the change in fluorescence, or lack thereof, will declare our victory or failure.