Team:BIT-China/Proof
Proof of concept
To prove our concept that we can make the bacteria sense the plasmid numbers and keep the numbers above a threshold, we separately designed two parts. One is to prove that plasmid numbers will influence the inhibitor concentration, the other is to prove the inhibitor concentration can adjust the expression of killer gene.
See our final results here!
Plasmid numbers will influence the inhibitor concentration
We use plasmids with different copies to adjust the plasmid numbers. To better simulate the real situation for application, we employed the constitutive promoter.
We built devices containing constitutive promoters with different strengths.
Meanwhile, we use RFP to replace the inhibitor gene to indicate the inhibitor concentration.
We have chosen plasmids with different copies and separately ligated these circuits on the vectors.
Through testing, we got data describing the relationship between the inhibitor concentration and plasmid copies.
This way, we verified that plasmid numbers will influence the inhibitor concentration.
Inhibitor concentration will adjust the expression of killer gene
We use arabinose induced promoter to control the expression of inhibitor protein. This way, through adding different concentration of arabinose, we can adjust the inhibitor concentration.
We built three devices containing different kinds of inhibitors.
Meanwhile, we built three corresponding in-promoter circuits and used RFP to replace the killer gene.
We assembled these corresponding circuits together for the final testing.
According to our expectation, the more arabinose added, the more inhibitor will be expressed. The downstream RFP will be repressed. That’s what we are going to prove.
But when we added the arabinose, the RFP intensity increased, and it contradicted with expected results. We thought the terminator cannot completely isolate the two devices.
So we change the promoter direction and add another B0015 to optimize the circuits.
We set an arabinose concentration gradient: 0, 0.0002％, 0.0005％, 0.0010％, 0.0015％, 0.0020％, 0.0030％, 0.0040％, 0.0050％. Under these concentrations, we measured the OD600 and RFP intensity.
The improvement of device construction that we added a terminator and changed the promoter direction did work and thus we observed the decrease of RFP intensity when we increase the arabinose concentration. It indicated that the change of arabinose concentration led to the change of inhibitor concentration, and the inhibitor can influence the expression of downstream gene. Here we use RFP gene to replace killer gene.
When the arabinose concentration reached to 0.0030％-0.0040％, the RFP can hardly express. The turning point proved that the inhibitor concentration at this point can almost completely repress the killer gene. Also, we proved that
inhibitor concentration can adjust the expression of killer gene.
Summary:
Above all, we proved that plasmid numbers will influence the concentration of inhibitor proteins, and the inhibitor concentration will influence the expression of killer gene which is indicated by RFP measurement results. This way, the change of plasmid numbers will influence the expression of killer gene, which means we can sense the plasmid numbers and accordingly decide whether or not to turn on the switch of killer gene.
