Team:BIT-China/Proof
To prove our concept that:
(1) We can make the bacteria sense the plasmid numbers.
(2) The in-promoter will respond differently to different signal which can reflect the plasmids losing on different levels. This way, our system can control the plasmids numbers above a threshold.
(2) The in-promoter will respond differently to different signal which can reflect the plasmids losing on different levels. This way, our system can control the plasmids numbers above a threshold.
We designed two parts. One of them is to prove that plasmid numbers will influence the inhibitor concentration. The other one is to prove the inhibitor concentration can regulate the expression of the killer gene by affecting its in-promoter.
Plasmid numbers will influence the inhibitor concentration
Because of the difficulty of controlling the number of plasmids, we can only choose some typical copy numbers of plasmids in our system.
The copy numbers are shown in the table below:
The copy numbers are shown in the table below:
Table.1 The copy numbers of different plasmids.
We also constructed the gene circuits containing constitutive promoters with
different strengths to express the inhibitor. By this we regulate the threshold
of plasmid numbers to meet different needs.
Fig.1 The device constructed to express the inhibitor with a constitutive promoter.
There are four promoters with different strengths and two kinds of RBS we have chosen:
Table.2 The strengths and efficiencies of different promoters and RBS.
Meanwhile, the RFP used to replace the inhibitor protein can directly represent the
concentration of the inhibitor in the cell. We separately constructed these circuits on different vectors which have different copy numbers.
At first, we use the modeling to explain the relationship of the concentration of the inhibitor and plasmids number like the curve below under a certain condition
In the wet experiment, after the same amount of time, we will measure the RFP intensity to get the data which can describe the relationship between the inhibitor concentration and the plasmid copy numbers.
The strength of J23106 is stronger J23116, we can clearly know the inhibitor protein have a positive correlation with plasmids number.
This way, we verified that plasmid numbers will influence the inhibitor concentration on the plasmid vector.
The inhibitor concentration can regulate the expression of the killer gene
In order to know the relationship between the inhibitor and in-promoter, we use the arabinose induced promoter PBAD to express the inhibitor. So we can add arabinose with different concentrations to induce the promoter and create an environment with different concentrations of intracellular inhibitor. Killer gene is replaced by RFP under control of in-promoter.
We built three devices containing different kinds of inhibitors. The gene circuits are shown in Fig.4.
Fig.4 Arabinose induced expression cassette of three kinds of inhibitors.
Meanwhile, we designed three corresponding in-promoter circuits in Fig.5.
Fig.5 In-promoter controlled expression cassette of RFP. RFP is used to replace killer gene.
We assembled these corresponding circuits together for the final testing.
Fig.6 Assembly of inhibitor and "in-promoter". They can be used to test the minimum arabinose concentration which can totally repress the expression of RFP.
We assumed that, more arabinose added, more inhibitor will be expressed and the downstream in-promoter will be repressed. That’s what we are going to prove.
But when arabinose was added, RFP intensity increased and it contradicted with the expected results. Maybe the terminator can’t completely isolate the two devices. Thought of it this way,
we change the promoter direction and add another B0015 to optimize the circuits.
The circuits are shown in the Fig.7.
So we change the promoter direction and add another B0015 to optimize the circuits.
So we change the promoter direction and add another B0015 to optimize the circuits.
Fig.7 Optimized circuits after changing the direction of promoter PBAD adding another terminator B0015.
After the pre-experiment, we chose a series of appropriate arabinose concentrations, and they are listed in Table.3. The negative control is the strain containing the empty vector pSB1C3 and the positive control is the strain containing the circuits mentioned above with no arabinose added.
Table.3 The concentrations of arabinose added in the experiment.
The improvement of device construction was that we added a terminator and changed the promoter direction. In this way, we could observe the decrease of RFP intensity when the arabinose concentration increases. It indicates that the change of arabinose concentration will affect inhibitor’s concentration, and the inhibitor can influence the expression of downstream gene. We chose the cI-Pr circuit to do this experiment and got the diagram describing the relationship between the time and RFP intensity under different concentration of arabinose.
Fig.8 RFP intensity measured under different concentration of arabinose.
From this diagram we can see, when the arabinose’s concentration reaches to 0.0030％-0.0040％, the RFP can hardly express. The result proved that the inhibitor can almost completely repress the killer gene at the turning point. Also,
we could say
inhibitor concentration can regulate the expression of the killer gene.
Summary:
Above all, we proved that plasmid numbers will influence the concentration of inhibitor proteins, and the inhibitor concentration will regulate the expression of killer gene which is indicated by RFP measurement results.
After connecting with killer gene, the plasmids losing on different levels 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. From all these, we can achieve the goal of controlling the number of plasmids as we need.
