PROOF OF CONCEPT

Our concept is based on three major devices that can be tested and optimised separately. Once they fulfill all of our strict criteria, they would be brought together to demonstrate our work. For an initial proof of concept of our work, we here present the three devices we decided suit our needs best.

AND GATE

This part takes nitric oxide (NO) and N-Acyl homoserine lactone (AHL) as its inputs and activates downstream gene expression. We were able to construct multiple versions of this AND gate (check out our Part Collection). After characterisation we decided on how to optimize it to best fit our purpose: detect the physiological concentration ranges of NO and AHL during inflammation.

We have chosen the variant that has shown the best dynamic range, BBa_K2116041, characterised through GFP expression. We could thus show that our first device is functional and works as expected (Figure 1).

Figure 1: Demonstration of AND gate behaviour in presence of NO and AHL. Measured 3 hours after induction. Error bars indicate S.D. of 3 technical replicates.. Our model shows that 4-fold activation would be sufficient for activating the next device; our switch.

SWITCH

Our switch consists of a recombinase, that irreversibly switches the reporter construct. Different recombinases were constructed: TP901, Bxb1. Here again, we focus on optimisation before assembly of all the parts into a larger circuit. We tested the recombinases codon optimised, non-optimized, with different RBSs as well as different degradation tags. Our favourite recombinase (codon optimized bxb1, BBa_K2116026) exhibits the most desired kinetic data (Figure 2). According to our model, some parameters of the switch (RBS strength and degradation rate of the integrase) must satisfy a specific ratio in order for the switch to work optimally. The kinetics shown in figure 2 show an ideal switching behavior, where steady state is reached quickly. Moreover in the 48 hours between transformation and measurements the leakiness of the tet promoter (0.05% according to our parameter estimation) was not enough to cause undesired flipping. This suggest that our switch can in principle store reliable information even for longer periods.

Figure 2: Demonstration of functionality and ideal flipping kinetics of bxb1 expressed under Ptet promoter. Bxb1 activation by aTc [ng/ml] and recombination mediated GFP expression. Data acquired using flow cytometry, error bars indicate SEM .

We conclude, that this switching rate is most desired for the whole construct.

REPORTER

Our final reporter consists of an AHL-inducible promoter flanked by recombinase specific att-sites. While not flipped, the reporter will express red fluorescent protein (mNectarine, BBa_K2116016). Upon flipping, mNectaring expression is expected to cease and GFP production to ensue. For proof of concept we tested our reporter with a constitutive promoter BBa_J23118 rather than an AHL-inducible promoter. Different reporter variants were tested in conjunction with our recombinases. We found that BBa_K2116024 is most suitable for our purposes, and works with our selected switch module.

PROOF OF CONCEPT SIMULATION

Figure 3 shows the functioning of the full system: Pavlov's Coli is encpsulated in a pill and is ingested by the patient. During transit in the gut the system is exposed to the inflammation marker (NO) and the candidate marker (species-specific AHL). In presence of both markers the promoters of the switch start flipping, storing the association event on the DNA. After recovery of the pill, the system is exposed to the candidate marker (AHL) again and since AHL was associated with an inflammation marker, GFP is expressed again. To learn more about the modelling of our system, see MODEL.

Figure 3: Simulation of the full system. Once NO and AHL are detected at the same time, our memory element start switching. After recovery of the pill, the system is exposed to AHL again and expresses GFP if NO and AHL were detected.

CONCLUSION

All three BioBrick devices were tested individually and display the desired function. The simulation of the full system shows that our system can work in principle, and the experimental data show that the switch is stable enough to store the detected information during the whole transit time through the gut. Thus we argue that the concept of associative learning with our genetic circuit has been proven. The system will be put together, and its function will be demonstrated under simulated real-world conditions (Demonstrate).