Team:IOANNINA/Engineering

Engineering

Overview Antibyeotic sensor Antibyeotic deactivation Kill Switch References

Overview

Synthetic Biology has a wide range of applications to everyday life. One aspect of SynBio is the modification of already existing microorganisms to have the desired characteristics that give them a useful function. The Engineering Cycle is the basis of building this idea and taking it one step closer to an applicable form.

General Engineering Cycle

Our project was based on the research and design of each of its modules as we didn’t have the necessary time to test all the modules that we designed. In many occasions we had to rethink or redesign specific aspects of our idea after contacting experts or exploring the bibliography further.

Design

As a team, we chose to tackle the problem of Antimicrobial Resistance (AMR) by using already existing AMR mechanisms, specifically antibiotic inactivating enzymes. We designed a living system that would degrade Tetracyclines and Macrolides in its environment (chicken manure) and then -for safety reasons- self-destruct. As a chassis organism, we chose the E. coli strain DH5a which is a common experimental model system.

Build

During the building stage, we separated our project in three modules: 1. Antibiotic sensor: The sensor consists of the Tetracycline and Macrolide binding repressor proteins, TetR and MphR. Based on the absence or presence of these antibiotics it will, or not, activate the kill switch. 2. Antibiotic deactivation: This module handles the inactivation of Tetracyclines and Macrolides, using the enzymes TetX2 and EreB respectively. 3. Kill switch: When antibiotics are absent, the kill switch is activated. It is based on the bpDNase1-mf-Lon toxin-antitoxin system designed by team IISER-Tirupati that we partnered with.

Test

Unfortunately, we did not have enough lab time to perform the needed experiments for our proof of concept. If we had managed to complete the construction of the final engineered organism, we would like to test its ability to deactivate antibiotics both in culture medium containing one or both of the targeted antibiotics, as well as poultry manure samples by measuring the amount of these antibiotics before and after the incubation.

Dry Lab Model

After research and with the help of the team IISER-Tirupati and Assistant Prof. Katapodis we tried to design the model that would fit our project the best. Team IISER-Tirupati mentored us by proposing some tools to get us started with the technical side of our modeling, while Assistant Prof. Katapodis helped us by pinpointing to us what kind of model we should deploy. Unfortunately, our desired model was heavily dependent on unavailable experimental data, thus not allowing us to complete the model of our system. The model we would deploy would be based on this article for our tetracycline insertion-neutralization cycle and this proposed model from iGEM Team TU-Munich for our macrolides insertion-neutralization cycle. Another important aspect we would model was the binding of tetracyclines and macrolides to the repressors as well as the kill switch regulation.

Learn

Hopefully, our design would have all the desired outcomes. However, this is not always the case. One of the most common problem is the inability to insert a functional gene on the bacteria. In that case, we should either modify our lab protocols or research for completely new ones. The other case is to come into a problem that concerns one or more of the three different modules. This is furtherly described later.

Antibiotic Sensor

To make our bacterial strain as safe as possible, we included a kill switch mechanism, as described above. The kill switch operates in such a way so that the strain may deactivate tetracyclines and macrolides in the presence of antibiotics but not in their absence. During the initial brainstorming period we came up with different methodologies to accomplish the kill switch’s regulation, from using some exogenous substance, to regulation of the system in a temperature dependent manner. After cycles of research and troubleshooting we concluded that the most efficient and affordable way to regulate this system would be by using the antibiotics themselves. In our research we were introduced to two gene regulators, TetR (Deng et al., 2013) and MphR (Cuthbertson & Nodwell, 2013), that can bind tetracyclines and macrolides respectively and can regulate the expression of downstream genes, in an antibiotic binding-dependent manner. The use of these two gene regulators would not require any extra steps to activate the kill switch mechanism and would be an easy and direct way of regulating the switch.

To build this antibiotic regulated system, we designed the necessary gene cassettes that included the two repressors, TetR and MphR with two different small tags to be able to detect the success of their expression. For this purpose, we built the basic parts BBa_K4056002 , BBa_K4056003 and the composite part BBa_K4056012.

Important questions that we would have to tackle with our dry lab work and the experimental procedures while testing the system: What modifications should we do to improve the antibiotic-binding sensitivity? Would the sensor functions in a similar manner when in the presence of only one of the selected antibiotics? Do TetR and MphR recognize other substances? Do TetR and MphR recognize the inactivated enzymes?

Antibiotic deactivation module

A crucial project decision was the choice of the target antibiotics. The final decision was taken based on factors such as: Which antibiotics are used in poultry the most? Which is the half-life of the antibiotics used? Which antibiotics are used in farming and humans too? What AMR mechanisms have evolved for the specific antibiotics? Which is their chemical composition? With the help of experts and bibliographic research, we decided on Tetracyclines and Macrolides. After this decision we had to design the deactivation system for both antibiotics. For this reason, we searched for the best antibiotic modifying enzymes available in naturally resistant bacterial strains that can modify irreversibly tetracyclines and macrolides respectively. Thus, we chose TetX2 for Tetracyclines (Besharati et al., 2019; Yang et al., 2004) and EreB for Macrolides (Morar et al., 2012). A key point was to design this module to be as safe as possible and to minimize the possibilities of horizontal gene transfer. For this reason, we decided to avoid the use of plasmid vectors for the final implementation. This module was designed to be integrated into the chromosome of the bacterial strain using the protocol of Cui and Shearwin (2017) to make sure that horizontal gene transfer doesn’t occur.

To build this module we needed to design the necessary genetic sequences and for this reason we built the basic parts BBa_K4056000, BBa_K4056001 and the composite parts BBa_K4056004, BBa_K4056002.

Once the gene modules were designed, we went ahead with the chromosomal integration in DH5a E.coli strains. The chromosomal integration of the TetX2 and EreB genes was tested by colony PCR as indicated by Cui and Shearwin’s clonetegration protocol.

Due to lack of time, we could not test the expression and activity of these enzymes. This test would be accomplished by an experimental procedure where we would incubate the bacteria in a culture medium that contained one or both of the targeted antibiotics. The survival of these bacteria would show that the enzymes are expressed and can function properly.

The chromosomal integration of the TetX2 and EreB genes was successful. However, if we had the necessary time to test the system, we would need to test the specific concentrations of antibiotics that these enzymes could deactivate and if the strain could function with both antibiotics present or if there would be an energy barrier. This learning procedure would give us insight on if this deactivation module that combines the deactivation of two antibiotics, could be used in our implementation proposal.

Kill Switch

The purpose of including the Kill Switch to our system was to ensure that the engineered bacteria will self-destruct once the antibiotics are deactivated. To design this kill switch, we initially thought of using a gene regulatory mechanism where we would use a repressor of the death gene regulatory element such as a gene repressor that would be regulated by TetR and MphR and would bind to the promoter of the death gene, thus repressing its expression when antibiotics were present. After discussions with team IISER-Tirupati, we decided to apply their designed protein kill switch that contained a bpDNase I as a toxin and mf-Lon protease as the antitoxin.

For the expression of these genes, we designed and built the basic part BBa_K4056007 composite parts BBa_K4056006 ,BBa_K4056010, BBa_K4056011, BBa_K405600214, BBa_K4056015 to use this toxin-antitoxin system in our application. To test in an initial stage the interaction of bpDNase I and mf-Lon we used an arabinose inducible promoter for bpDNase I to avoid the bacterial self-destruction before we test the interaction.

To test the efficiency of the antibiotic sensor - kill switch system we should cultivate our engineered bacteria in a culture that contains either Tetracyclines or Macrolides or both. The survival of the engineered bacteria will prove the ability of the sensor and mf-Lon to prevent cell death. Then the colonies should be transferred in an antibiotic-free culture medium. No colonies should survive there, proving that bpDNase I induces death and the sensor is working.

This final testing step would be a learning procedure as we would find solutions to the problems that would occur in each stage before testing the final proof of concept.