iGEM 2021 — Team IOANNINA

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# Team:IOANNINA/Notebook

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[Design](https://2021.igem.org/Team%3AIOANNINA/Design)
[Notebook](https://2021.igem.org/Team%3AIOANNINA/Notebook)
[Protocols](https://2021.igem.org/Team%3AIOANNINA/Protocols)
[Safety](https://2021.igem.org/Team%3AIOANNINA/Safety)

# Notebook

[Week 1](#week1)
[Week 2](#week2)
[Week 3](#week3)
[Week 4](#week4)
[Week 5](#week5)
[Next Steps](#nextsteps)
[References](#refs)

### Week 1 (10-16/9) : Preparation of plasmid pSB1C3

The first step was to get familiar with the experimental procedures and make our first transformation of the plasmid pSB1C3-RFP, provided in the distribution kit.

* After plamid pSB1C3-RFP was dissolved from distribution kit we transformed the high efficiency competent E. coli DH5a strains, provided by NEB. Growth of the cells happened at agar plates ( + Chloramphenicol) overnight at 37°C, giving the next day succesful results. We chose specific colonies and made precultures of pSB1C3-RFP (Vfinal=3ml) to isolate the pSB1C3-RFP plasmid with the Monarch Plasmid Mini-prep Kit.
* The plasmids were digested with EcoRI to check that the isolated plasmid was the correct one and once we were sure, we made glycerol stocks of the cultures that contained the plasmid.
* By the end of the first week, we prepared everything needed for the next one, specifically agar plates and LB for bacteria cultures (LB-Agar + Kan, LB-Agar + Amp).

### Week 2 (20-24/9) : Preparation of pOSIP-KO and pE-FLP plasmids

We received the two plasmids that we needed for the chromosomal integration of two of our genes according to the Clonetegration protocol by (Cui and Shearwin, 2017). The plasmids were pOSIP-KO and pE-FLP provided by Addgene and arrived as bacterial agar stabs.

* We applied a streaking method using sterile toothpicks in order to isolate the bacteria containing the plasmids and plated them in agar plates containing the required antibiotics. The culture plates were incubated at 30°C, as at higher temperatures the integrase gene is expressed thus leading to integration of the plasmid sequence into the chromosome. This would make impossible the isolation of the plasmid for a cloning procedure.
* The next day and after the bacteria had grown in the plate, we made precultures of selected colonies and proceeded to miniprep the next day to isolate the plasmids. To check the efficiency of the miniprep we did an electrophoresis which indicated that we had none to very small concentration of both plasmids. We repeated the miniprep procedure but the same results occured again.
* This was an indication that something went wrong in some stage of the procedure. As this system is temperature-dependent, we supposed that there could have been some accidental arisal of the temperature in some stage, resulting in chromosomal integration before the plasmid isolation. For the next week, we decided to make a colony PCR as described by Cui and Shearwick in their clonetegration protocol, to check for this possibility.
* Also, in this week we received the antibiotic deactivation module by IDT, which was resuspended and ready for use once the pOSIP-KO plasmid was ready.

### Week 3 (27/9-1/10): Troubleshooting

* Due to the minimal concentration of pOSIP-KO & pE-FLP plasmids, we proceeded with a colony PCR to check for possible chromosomal integration. This process did not give clear results, but indicated that our initial suggestion was right. We repeated the colony PCR and had a strong indication that the chromosomal integration had happened.
* For this reason, and due to the pressure of time we decided to restreak the initial agarose stab, starting in the end of this week.

### Week 4 (5-7/10) : Isolation and purification of pOSIP-KO plasmid

* This week we went on restreaking the initial agarose stabs and repeating the process of plating, making precultures and minipreps. The electrophoresis that took place at the end of the week showed that we had successfully isolated and cleaned the desired plasmids. To make sure we had the correct plasmids, we digested a small amount of the plasmid with EcoRI to check for the length of the DNA sequence.
* To proceed with the HiFi DNA Assembly by NEB, we needed to linearize our vector pOSIP-KO through EcoRI and PstI digestion. To make sure that the two enzymes were fully functional, we tested them in different enzyme buffers (as their initial ones didn’t match) and decided on which combination gave the best result.

### Week 5 (11-15/10) : Assembly of pOSIP-KO/TetX2-EreB and Clontegration

* We were now finally ready to assemble our first gene cassette into the pOSIP-KO plasmid vector.

First, we did a digestion reaction with 2.5 μg of pOSIP-KO with the restriction enzymes PstI and EcoRI. Due to some problems during the digestion procedure, we were not able to proceed with the assembly. The digestions were repeated the next day.- The digestion of the plasmid vector was repeated and as the electrophoresis that followed showed, the linearized plasmid was ready for the assembly reaction

NEB HiFi DNA Assembly reaction

* After quantification of both the linearised plasmid and the insert (antibiotic deactivation module) we ended up using a ration of vector: insert of 1:2 and specifically 0.04 pmoles of insert and 0.02 pmoles of vector in the NEB Hifi DNA Assembly reaction.

Transformation-Clonetegration

* After the assembly, we transformed NEB high efficiency competent E.coli DH5a strains and plated for the control reaction ampicillin plates and for the assembled pOSIP-KO plasmid Kanamycin plates with a lower concentration of antibiotics than normally used (we used 20ul/ml).
* In the 1 h incubation of the transformed cells at 37°C, the integrase was produced thus leading to the chromosomal integration of the plasmid that contained the deactivation module.
* The next day we observed colonies that were growing in a slow rate, however after 2 days of incubation in 30°C, full size colonies were picked.
* We performed a colony PCR that showed the success of our clonetegration.

This week we also received the pOSIP-KH plasmid vector that will be used for the chromosomal integration of the other modules of our project into the chromosome. However, due to lack of time, it is possible that we will not achieve this goal until the Wiki Freeze.

### Proposed Experiments for the next steps

Even though we didn’t have the desired time to finish all our experiments, we have done a design of the next steps needed to have a full proof of concept of our lab design.

Test of Deactivation Module

As we have already completed the first chromosomal integration of the antibiotic deactivation module, next step would be testing its efficiency. For this, different amounts of tetracycline and erythromycin (macrolide) would be needed to check which concentration can be deactivated in an efficient manner.

Test of kill switch

To test the kill switch that was designed by IISER-Tirupati, a plasmid construct of pSB1C3, containing the kill switch gene cassette would be cloned by the NEB Hifi assembly protocol and transformed into DH5a competent cells, in the presence of arabinose. The bacterial OD would be measured to check if the bacteria can live while mf-lon gene is expressed. An extra plasmid containing only the gene of bpDNase I under the control of an arabinose inducible promoter could be used as a control.

Final bacterial strain construction

If the testing of the kill switch gives the desired results, we could proceed with the chromosomal integration of the bpDNase I, using a second plasmid, pOSIP-KH, which would allow the insertion of the gene cassette in a different position of the chromosomal DNA than the one already used (pOSIP-KO).The gene cassette of the bpDNase I could be assembled with the antibiotic sensor module and integrated into the chromosomal DNA as one sequence. Mf-lon gene would be cloned in a pSB1C3 plasmid in two copies, the first one being under the control of mphO operator and the other one under the control of tetO operator.

Test of antibiotic sensor-Proof of concept

Once this bacterial strain is constructed, different concentrations of tetracycline and erythromycin would be used to check the efficiency of the antibiotic sensor module. The results of this experiment would be counted as the bacterial OD. In case the sensor works, bacteria would live or self-destruct depending on the presence or absence of the antibiotics. This would give the final proof of concept of our design.

### References

[Cui, L., Shearwin, K.E., 2017. Clonetegration Using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria, in: Hughes, R.A. (Ed.), Synthetic DNA: Methods and Protocols, Methods in Molecular Biology. Springer, New York, NY, pp. 139–155.](https://doi.org/10.1007/978-1-4939-6343-0_11)

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# Team:IOANNINA/Engineering

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[Implementation](https://2021.igem.org/Team%3AIOANNINA/Implementation)
[Engineering](https://2021.igem.org/Team%3AIOANNINA/Engineering)
[HumanPractices](https://2021.igem.org/Team%3AIOANNINA/Human_Practices)
[Communication](https://2021.igem.org/Team%3AIOANNINA/Communication)
[Sustainable](https://2021.igem.org/Team%3AIOANNINA/Sustainable)
[Design](https://2021.igem.org/Team%3AIOANNINA/Design)
[Notebook](https://2021.igem.org/Team%3AIOANNINA/Notebook)
[Protocols](https://2021.igem.org/Team%3AIOANNINA/Protocols)
[Safety](https://2021.igem.org/Team%3AIOANNINA/Safety)

# Engineering

[Overview](#over)
[Antibyeotic sensor](#sensor)
[Antibyeotic deactivation](#deact)
[Kill Switch](#switch)
[References](#refs)

### 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](https://pubs.acs.org/doi/10.1021/bi702296k) article for our tetracycline insertion-neutralization cycle and [this](https://2013.igem.org/Team%3ATU-Munich/Modeling/Enzyme) 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.

### References

[Besharati, M., Hamedi, J., Hosseinkhani, S., Saber, R., 2019. A novel electrochemical biosensor based on TetX2 monooxygenase immobilized on a nano-porous glassy carbon electrode for tetracycline residue detection. Bioelectrochemistry 128, 66–73.](https://doi.org/10.1016/j.bioelechem.2019.02.010)
[Cui, L., & Shearwin, K. E. (2017). Clonetegration Using OSIP Plasmids: One-Step DNA Assembly and Site-Specific Genomic Integration in Bacteria. Methods in molecular biology (Clifton, N.J.), 1472, 139–155.](https://doi.org/10.1007/978-1-4939-6343-0_11)
[Deng, W., Li, C., Xie, J., 2013. The underlying mechanism of bacterial TetR/AcrR family transcriptional repressors. Cellular Signalling 25, 1608–1613.](https://doi.org/10.1016/j.cellsig.2013.04.003)
[Morar, M., Pengelly, K., Koteva, K., Wright, G.D., 2012. Mechanism and diversity of the erythromycin esterase family of enzymes. Biochemistry 51, 1740–1751.](https://doi.org/10.1021/bi201790u%20)
[Yang, W., Moore, I.F., Koteva, K.P., Bareich, D.C., Hughes, D.W., Wright, G.D., 2004. TetX Is a Flavin-dependent Monooxygenase Conferring Resistance to Tetracycline Antibiotics \*. Journal of Biological Chemistry 279, 52346–52352.](https://doi.org/10.1074/jbc.M409573200)
[Zheng, J., Sagar, V., Smolinsky, A., Bourke, C., LaRonde-LeBlanc, N., Cropp, T.A., 2009. Structure and Function of the Macrolide Biosensor Protein, MphR(A), with and without Erythromycin. Journal of Molecular Biology 387, 1250–1260.](https://doi.org/10.1016/j.jmb.2009.02.058)

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# Team:IOANNINA/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

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# Team:IOANNINA/Results

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[Engineering](https://2021.igem.org/Team%3AIOANNINA/Engineering)
[HumanPractices](https://2021.igem.org/Team%3AIOANNINA/Human_Practices)
[Communication](https://2021.igem.org/Team%3AIOANNINA/Communication)
[Sustainable](https://2021.igem.org/Team%3AIOANNINA/Sustainable)
[Design](https://2021.igem.org/Team%3AIOANNINA/Design)
[Notebook](https://2021.igem.org/Team%3AIOANNINA/Notebook)
[Protocols](https://2021.igem.org/Team%3AIOANNINA/Protocols)
[Safety](https://2021.igem.org/Team%3AIOANNINA/Safety)

# 2021 Jamboree Results

### Bronze Medal

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### Rookie Judge Prize

Lefkothea-Vasiliki Andreou

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