iGEM 2021 — Team Leiden

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# Team:Leiden/Notebook

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DOPLLOCK iGEM Leiden

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Project

[Description](https://2021.igem.org/Team%3ALeiden/Description)
[Proof-of-Concept](https://2021.igem.org/Team%3ALeiden/Proof_Of_Concept)
[Engineering](https://2021.igem.org/Team%3ALeiden/Engineering)
[Results](https://2021.igem.org/Team%3ALeiden/Results)
[Model](https://2021.igem.org/Team%3ALeiden/Model)
[Contribution](https://2021.igem.org/Team%3ALeiden/Contribution)
[Measurement](https://2021.igem.org/Team%3ALeiden/Measurement)
[Kit-Search](https://2021.igem.org/Team%3ALeiden/Kit-Search)

Lab

[Parts](https://2021.igem.org/Team%3ALeiden/Parts)
[Notebook](https://2021.igem.org/Team%3ALeiden/Notebook)
[Safety](https://2021.igem.org/Team%3ALeiden/Safety)
[Experiments](https://2021.igem.org/Team%3ALeiden/Experiments)

Human Practices

[Integrated Human Practices](https://2021.igem.org/Team%3ALeiden/Human_Practices)
[Public Engagement](https://2021.igem.org/Team%3ALeiden/Communication)
[Education](https://2021.igem.org/Team%3ALeiden/Education)
[Entrepreneurship](https://2021.igem.org/Team%3ALeiden/Entrepreneurship)
[Implementation](https://2021.igem.org/Team%3ALeiden/Implementation)
[Survey](https://2021.igem.org/Team%3ALeiden/Survey)

Team

[Team Members](https://2021.igem.org/Team%3ALeiden/Team)
[Attribution](https://2021.igem.org/Team%3ALeiden/Attributions)
[Collaborations](https://2021.igem.org/Team%3ALeiden/Collaborations)
[Sponsors](https://2021.igem.org/Team%3ALeiden/Sponsors)

Outreach

[Featured](https://2021.igem.org/Team%3ALeiden/Featured)
[Newsletter](https://2021.igem.org/Team%3ALeiden/Newsletter)
[Crowdfunding](https://2021.igem.org/Team%3ALeiden/Crowdfunding)

☰

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# Notebook

Below you can find the notebook of the experiments that we have performed in chronological order. The experiments are categorized in three research lines that we have worked on: 1. DOPL LOCK system, 2. Genome integration, and 3. Improve-a-part. A more brief overview of the experiments that we have performed can be found on the [Experiments page](https://2021.igem.org/Team%3ALeiden/Experiments). In addition, an elaborated description of the protocols that we have used can also be found there. Furthermore, a full clear overview of the results can be found on the [Results page](https://2021.igem.org/Team%3ALeiden/Results). The notebook was written and exported with Benchling.

[x]

* # DOPL LOCK system

[x]

* # Genome integration

[x]

* # Improve-a-part

#### Find Out More

* [Description](https://2021.igem.org/Team%3ALeiden/Description)
* [Proof of Concept](https://2021.igem.org/Team%3ALeiden/Proof_Of_Concept)
* [Implementation](https://2021.igem.org/Team%3ALeiden/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3ALeiden/Entrepreneurship)
* [Integrated Human Practices](https://2021.igem.org/Team%3ALeiden/Human_Practices)

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©2021 All rights reserved.

#### Contact Us

* Email
* igem@science.leidenuniv.nl
* Address
* Sylviusweg 72, 2333BE Leiden
  the Netherlands

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# Team:Leiden/Engineering

<!DOCTYPE html>

DOPLLOCK iGEM Leiden

Home

Project

[Description](https://2021.igem.org/Team%3ALeiden/Description)
[Proof-of-Concept](https://2021.igem.org/Team%3ALeiden/Proof_Of_Concept)
[Engineering](https://2021.igem.org/Team%3ALeiden/Engineering)
[Results](https://2021.igem.org/Team%3ALeiden/Results)
[Model](https://2021.igem.org/Team%3ALeiden/Model)
[Contribution](https://2021.igem.org/Team%3ALeiden/Contribution)
[Measurement](https://2021.igem.org/Team%3ALeiden/Measurement)
[Kit-Search](https://2021.igem.org/Team%3ALeiden/Kit-Search)

Lab

[Parts](https://2021.igem.org/Team%3ALeiden/Parts)
[Notebook](https://2021.igem.org/Team%3ALeiden/Notebook)
[Safety](https://2021.igem.org/Team%3ALeiden/Safety)
[Experiments](https://2021.igem.org/Team%3ALeiden/Experiments)

Human Practices

[Integrated Human Practices](https://2021.igem.org/Team%3ALeiden/Human_Practices)
[Public Engagement](https://2021.igem.org/Team%3ALeiden/Communication)
[Education](https://2021.igem.org/Team%3ALeiden/Education)
[Entrepreneurship](https://2021.igem.org/Team%3ALeiden/Entrepreneurship)
[Implementation](https://2021.igem.org/Team%3ALeiden/Implementation)
[Survey](https://2021.igem.org/Team%3ALeiden/Survey)

Team

[Team Members](https://2021.igem.org/Team%3ALeiden/Team)
[Attribution](https://2021.igem.org/Team%3ALeiden/Attributions)
[Collaborations](https://2021.igem.org/Team%3ALeiden/Collaborations)
[Sponsors](https://2021.igem.org/Team%3ALeiden/Sponsors)

Outreach

[Featured](https://2021.igem.org/Team%3ALeiden/Featured)
[Newsletter](https://2021.igem.org/Team%3ALeiden/Newsletter)
[Crowdfunding](https://2021.igem.org/Team%3ALeiden/Crowdfunding)

☰

[![DOPL LOCK](https://static.igem.org/mediawiki/2021/9/9c/T--Leiden--whitelogo.png)](https://2021.igem.org/Team%3ALeiden)

# engineering

In this page we elaborate how we have used the Design-Build-Test-Learn cycle to design and improve different aspects of DOPL LOCK. We have used this engineering principle to determine the compatibility of different origins of replication (Ori) and determine the efficacy of multiple bacterial toxin-antitoxins systems. How the final version of DOPL LOCK will look is described in the [Proof of concept](https://2021.igem.org/Team%3ALeiden/Proof_Of_Concept) page.

* [Engineering success](#Engineering_success)
* [Double Plasmid](#Double_Plasmid)
* [Toxins](#Toxins)

# Engineering success

One great tool for the successful engineering of genetic circuits is the Design-Build-Test-Learn (DBTL) cycle (Figure 1). This strategy leverages engineering principles and provides an efficient, generalized and highly systematic framework for the engineering of complex genetic circuits. **Design** principles are used to specify a biological system with an intended function. The desired DNA sequence encoding the biological system was **built** and implemented into our target organism. The functionality of our biological system has been **tested**. We have learned from the divergence between the expected and measured functionality of our system to improve the initial design.

![Gold medal](https://static.igem.org/mediawiki/2021/b/b7/T--Leiden--Engeneering--DBTLcycle.png)

*Figure 1: The Design-Build-Test-Learn cycle*

In our project, we have adapted this tool to design the different components of our DOPL LOCK system. We have used the DBTL cycle (i) to determine which two **origins of replication (Ori)** were most compatible for our system and (ii) which of the **toxin-antitoxin systems** will show the best performance regarding inducing **cell death.**

# Double Plasmid

Two of the most commonly used methods for maintaining **recombinant DNA** in an organism are the **integration** of the DNA in the **genome** and maintaining the recombinant DNA on an independently replicating **plasmid** [1]. Keeping biosafety in mind, **plasmids** have advantages over genome integration since (i) it is **easier** to be introduced in the cell, (ii) it is more easy to control the **amount of copies**, and (iii) most importantly there is very little to no **homology** between the **plasmid** DNA and the **genome** of the host bacterium and potentially other environmental bacteria [2]. This would **reduce** the likelihood of **horizontal gene transfer (HGT)** drastically, unless the plasmid is transferred completely [3].

## Research and imagine

To create a double plasmid system we would need to have stable maintenance of the **two plasmids** in one bacterium. Previous research has shown that co-transforming two plasmids with the same **incompatibility group** will lead to **plasmid segregation** and **unwanted recombination events** [4]. This could result in one plasmid outcompeting the other **unstabilizing** the system. Additionally, the two plasmids could recombine in one plasmids increasing the risk of horizontal gene transfer (HGT).

The plasmid incompatibility is determined by the **Ori** of the two plasmids, which is the starting point for the amplification of the plasmid DNA. When two plasmids require the same **replication mechanism**, they belong to the same **incompatibility group** [5]. There are over 27 incompatibility groups represented in the *Enterobacteriaceae* family and this is expanding since new genomic data is generated [6].

## Design and Build

In our project, **we will use two plasmids with compatible Oris.** We will design our system in such a way that the plasmids we use have different incompatibility groups and therefore will be **stably maintained** in the cell. Our system will be based on the **pJUMP plasmids** [7] obtained via Addgene. These plasmids are designed according to the **Standard European Vector Architecture** (SEVA) nomenclature [8]. We have been testing which plasmids are most compatible for our system by **co-transforming** two plasmids with various **Oris**, different **fluorophores**, and different **selection markers** as shown in Figure 2. It is expected that upon co-transforming two compatible plasmids the expression of GFP and RFP is **comparable** to when the plasmids are transformed **solely**.

![Design and Build](https://static.igem.org/mediawiki/2021/thumb/9/96/T--Leiden--Engeneering--GFP_RFP_Constructs.png/706px-T--Leiden--Engeneering--GFP_RFP_Constructs.png)

*Figure 2: Schematic visualization of the co-transformation assay where two plasmids with GFP and RFP are transformed in one bacterium.*

The plasmids we use for our system are derived from the lab of Christopher French and are shown in Table 1 [7].

The plasmids

*Table 1: The different JUMP plasmids used in our project.*

| NAME | RESISTANCE | ORIGIN OF REPLICATION | INSERT | INCOMPATIBILITY GROUP | COPY NUMBER |
| --- | --- | --- | --- | --- | --- |
| pJUMP26 | Kanamycin | p15A | sfGFP | B | Medium/Low copy number |
| pJUMP27 | Kanamycin | pSC101 | sfGFP | C | Low copy number |
| pJUMP28 | Kanamycin | pUC | sfGFP | A | High copy number |
| pJUMP29 | Kanamycin | pBR322/ROB | sfGFP | A | Medium/High Copy number |
| pJUMP46 | Spectinomycin | p15A | RFP | B | Medium/Low copy number |
| pJUMP47 | Spectinomycin | pSC101 | RFP | C | Low copy number |
| pJUMP48 | Spectinomycin | pUC | RFP | A | High copy number |
| pJUMP49 | Spectinomycin | pBR322/ROB | RFP | A | Medium/High Copy number |

To be able to determine which of the plasmids are maintained in the cell at similar levels we switched the sfGFP cassette of the pJUMP40 plasmids to a RFP cassette ([BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450)) according to [Experiment 1: sfGFP-RFP swap](https://2021.igem.org/Team%3ALeiden/Experiments#Experiment 1: sfGFP-RFP swap). Both vectors are co-transformed in *Escherichia coli* TOP10 according to [Experiment 2: (Co-)transformation of sfGFP and RFP plasmids](https://2021.igem.org/Team%3ALeiden/Experiments#Experiment 2: (Co-)transformation of sfGFP and RFP plasmids).

## Test and Learn

To test which of the plasmids are **stably maintained** in the cell after **co-transformation**, the green and red **fluorescence signal** is measured according to [Experiment 5: Plate reader fluorescence assay](https://2021.igem.org/Team%3ALeiden/Experiments#Experiment 5: Plate reader fluorescence assay). The data of 3 biological replicates was analysed using R-studio and is presented in Figure 3 and are further elaborated in the [Results](https://2021.igem.org/Team%3ALeiden/Results).

![](https://static.igem.org/mediawiki/2021/f/f2/T--Leiden--Engeneering--Doubleplasmid.png)

*Figure 3: Fluorescence presented of the different co-transformed E. coli TOP10 strains.*

When the engineered pJUMP plasmids were **solely** transformed in *E. coli* TOP10 we observed that the appropriate fluorescent markers were expressed. However, for pJUMP49 we observed some signal in the GFP emission channel (510 nm), while this should not be the case.

When the plasmids were **co-transformed** in different combinations to determine which combination of Oris are most suitable for co-transformation, it was found that pJUMP26 replicating via the Ori **p15A** and pJUMP49 which replicates via the Ori **pBR322/ROB**, showed **similar** levels of fluorescence of both **fluorescent markers**. Therefore, our data shows us that the Oris **p15A** and **pBR322/ROB** seem most compatible for applications where both plasmids need to express recombinant proteins at **similar levels** within the cell.

## Redesign

For our project, we have decided that we would make a first version of the DOPL LOCK system in plasmids with the Oris **p15A and pBR322/ROB** due to the fact that they are maintained in similar levels and use **different replication mechanisms** to replicate the plasmid. These features reduce unwanted effects, such as plasmid recombination, and enable us to utilize both plasmids to full potential. Additionally, we would like to test a new series of plasmids which are compatible with SEVA, Biobricks and Type IIS restriction enzymes. This provides us with **more flexibility** since these SEVA plasmids are more compatible with **different cloning methods** [9]. Additionally, the SEVA 3.1 plasmids enable us to test more efficiently since the cloning is **reversible** [9]. Additionally, we would use the SEVA plasmid's **modularity** to our advantage to test more different Oris. This will expand our **toolset** of compatible Ori's and flexibility when **tailoring** our system to fulfill the need of a **specific application**.

# Toxins

Just in case **HGT** occurs, we want this to be as **disadvantageous** as possible for the recipient bacterium. This way we would **prevent** further spread of the **synthetic genes** throughout nature. Due to the fact that the DNA is disadvantageous, the bacterium will most likely **discard** the DNA or will be **outcompeted** by the other bacteria. One way of creating this disadvantage is by letting the plasmid produce a **toxin**. This toxin will most likely kill the bacterium and therefore never be able to outcompete the bacteria present in the environment. We used the **DBTL cycle** to design genetic circuits to test the efficacy of the toxins, as described below. The **efficacy** of the toxin is determined by measuring the **OD600** over the timespan of 16h after inducing the toxin. Subsequently, we used this data to evaluate which toxins seem **most suitable** to incorporate in our DOPL LOCK system.

## Research and imagine

When selecting possible **candidate toxins** for the DOPL LOCK system, we wanted the toxin to **kill** the bacteria instead of only eliminating growth. Additionally, the ability to neutralize the toxic effect via the expression of an **antitoxin** would be favoured. In literature, we found that multiple toxins posses this criterium, namly the **RelE/RelB** TA-system [10], the **MazF/MazE** TA-system [11], the **HOK/SOK** TA-system [12] and the **CcdB/CcdA** TA-system [13]. Furthermore, we uploaded the DNA sequences form the TA systems in benchling and extracted the sequences from the toxins and antitoxins, and created two separate sequences. These DNA sequences were synthesized and assembled in various **genetic circuits.**

## Design and build

To test the **efficacy** of the toxins we have picked for our system, we designed a **genetic circuit** that enables us to **control** the expression of the toxin. This genetic circuit is composed out of the **pBAD** promoter ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)), a **ribosomal binding site** (RBS) ([BBa\_J34801](http://parts.igem.org/Part%3ABBa_J34801)), the **coding sequences of the toxins** and a **lambda T1 terminator** ([BBa\_K864601](http://parts.igem.org/Part%3ABBa_K864601)), as shown in Figure 4.

![PromotorToxinConstructs](https://static.igem.org/mediawiki/2021/3/3c/T--Leiden--Engeneering--PromotorToxinConstructs.png)

*Figure 4: Schematic representation of the architecture of the different constructs for testing toxin efficacy.*

To enable the construction of our genetic circuits, we obtained the standardized parts and coding sequences for the **toxins** as gBlock DNA from our valued sponsor **Integrated DNA technologies.** The gBlock DNA is dissolved and cloned into the pSB1A3 and pSB1C3 plasmid backbones according to [E](https://2021.igem.org/Team%3ALeiden/Experiments)[xperiment](https://2021.igem.org/Team%3ALeiden/Experiments) [4](https://2021.igem.org/Team%3ALeiden/Experiments). Subsequently, the genetic circuit is assembled using 3A-assembly according to [Experiment 3: Biobrick ligation](https://2021.igem.org/Team%3ALeiden/Experiments#Experiment 3: Biobrick ligation) in the plasmid pJUMP26 [6]. These vectors were transformed in the *E. coli* TOP10 strain according to [Experiment 4: 3A assembly](https://2021.igem.org/Team%3ALeiden/Experiments#Experiment 4: 3A assembly).

## Test and learn

To test the potency of our toxins, transformed *E. coli* bacteria were treated according to [Experiment 6: Toxicity assay in liquid medium](https://2021.igem.org/Team%3ALeiden/Experiments#Experiment 6: Toxicity assay in liquid medium) to start toxin production. Subsequently, the OD600 is measured for 16h using a Tecan Spark microtiter plate reader. The data of 4 biological replicates was analysed using R-studio and shown in Figure 5.

![pBAD Toxin](https://static.igem.org/mediawiki/2021/thumb/0/0b/T--Leiden--Engeneering--pBADtoxin.png/484px-T--Leiden--Engeneering--pBADtoxin.png)

*Figure 5: Induction of the HOK, MazF and RelE toxins via supplementation of different amounts of arabinose and the effect on the OD600 over a time span of 16 hours.*

Our data shows that, when the toxin expression is induced the cells do not immediately die but their **growth rate is reduced**. Additionally, the more inducer is present in the medium, the more the growth rate is reduced. However, during our [human practices](https://2021.igem.org/Team%3ALeiden/Human_Practices) meeting with **Nathan Fraikint** we discussed the data and he advised us to test the toxin efficiency on LB **agar plates** and not in **liquid LB medium**. Supposedly, in liquid media the bacteria that escape the kill switch can easily **overgrow** the bacteria affected by **the toxin** [14]. Therefore, we decided to develop an **alternative protocol** ([Experiment 7: Toxicity assay by LB-agar plate](https://2021.igem.org/Team%3ALeiden/Experiments#Experiment 7: Toxicity assay by LB-agar plate)) for testing the efficacy of the **toxins**. This was used to determine the toxicity of the toxins.

Additionally, when discussing our experiments with **Prof. Dr. Dennis Claessen**, he advised us to use a **minimal medium** instead of **LB medium** to test the toxin efficiency. This would be more representative due to the fact that in a situation where the organism **escapes** into the environment, nutrients would also be a **limiting factor**. Therefore, when using minimal medium we could get a more realistic effect of the toxicity of the toxins in a **representative situation**.

Finally, when we revisited literature to find possible explanations for the lack of lethality of the toxins, we found that in similar studies, researchers used the *E. coli* strain **MC4100** to test the efficacy of the MazF toxin [15]. This strain has a non-functional ***relA1*** **gene**, which is responsible for the synthesis of ppGpp, a key signal molecule in the prokaryotic stress response. When this gene is absent, the general stress response is not initiated by the expression of the toxin, therefore the *E. coli* strain MC4100 seems a better candidate for testing the efficacy of the toxins. This strain could **potentially** be interesting to use in our system, since it seems **more susceptible to stress** induced by our toxins. Additionally, we use the *E. coli* strain MC4100 as a **potential host** for our DOPL LOCK system.

## Re-design

For further testing of our system, we have planned to transform constructs that express the toxins in the *E. coli* strains **TOP10 and MC4100**, and grow these in M9 minimal medium as suggested by **Prof. Dr. Dennis Claessen**. Additionally, we have developed a new protocol to determine the efficiency of the toxins on solid LB agar plates. This will give us insights into the lethality of the system in more representative conditions and what the effect of the ppGpp pathway is on the probability of **escaping our mechanism**.

Beside altering the **testing procedure,** we would also like to change the design of our toxin cassettes. We would like to test if **multiple copies of the toxin expression cassette** results in more effective induction of cell death, and if it does how many copies are **optimal**. Additionally, we would like to test if the antitoxin is able to **neutralize the toxic effect** of the toxin when expressed from **another plasmid**. Finally, we would like to investigate if more **exotic toxins** also work in our system, expanding the toolbox and flexibility when tailoring our system to fulfill the need of a **specific application**.

# References

1. Kroll, J., Klinter, S., Schneider, C., Voss, I., & Steinbüchel, A. (2010). Plasmid addiction systems: perspectives and applications in biotechnology. Microbial biotechnology, 3(6), 634–657. https://doi.org/10.1111/j.1751-7915.2010.00170.x
2. Wright, O., Delmans, M., Stan, G. B., & Ellis, T. (2015). GeneGuard: A modular plasmid system designed for biosafety. *ACS synthetic biology*, *4*(3), 307–316. https://doi.org/10.1021/sb500234s.
3. Harrison, E., & Brockhurst, M. A. (2012). Plasmid-mediated horizontal gene transfer is a coevolutionary process. Trends in microbiology, 20(6), 262–267. https://doi.org/10.1016/j.tim.2012.04.003
4. Novick R. P. (1987). Plasmid incompatibility. Microbiological reviews, 51(4), 381–395. https://doi.org/10.1128/mr.51.4.381-395.1987
5. del Solar, G., Giraldo, R., Ruiz-Echevarría, M. J., Espinosa, M., & Díaz-Orejas, R. (1998). Replication and control of circular bacterial plasmids. *Microbiology and molecular biology reviews : MMBR*, *62*(2), 434–464. https://doi.org/10.1128/MMBR.62.2.434-464.1998
6. Shintani, M., Sanchez, Z. K., & Kimbara, K. (2015). Genomics of microbial plasmids: classification and identification based on replication and transfer systems and host taxonomy. *Frontiers in microbiology*, *6*, 242. https://doi.org/10.3389/fmicb.2015.00242
7. Valenzuela-Ortega M, French CE. Joint Universal Modular Plasmids: A Flexible Platform for Golden Gate Assembly in Any Microbial Host. Methods Mol Biol. 2020;2205:255-273. doi: 10.1007/978-1-0716-0908-8\_15. PMID: 32809204.
8. Silva-Rocha, R., Martínez-García, E., Calles, B., Chavarría, M., Arce-Rodríguez, A., de Las Heras, A., Páez-Espino, A. D., Durante-Rodríguez, G., Kim, J., Nikel, P. I., Platero, R., & de Lorenzo, V. (2013). The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes. *Nucleic acids research*, *41*(Database issue), D666–D675. https://doi.org/10.1093/nar/gks1119
9. Damalas, S. G., Batianis, C., Martin-Pascual, M., de Lorenzo, V., & Martins Dos Santos, V. (2020). SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial biotechnology, 13(6), 1793–1806. https://doi.org/10.1111/1751-7915.13609
10. Overgaard, M., Borch, J., & Gerdes, K. (2009). RelB and RelE of Escherichia coli form a tight complex that represses transcription via the ribbon-helix-helix motif in RelB. *Journal of molecular biology*, *394*(2), 183–196. https://doi.org/10.1016/j.jmb.2009.09.006
11. Yeo, C. C., Abu Bakar, F., Chan, W. T., Espinosa, M., & Harikrishna, J. A. (2016). Heterologous Expression of Toxins from Bacterial Toxin-Antitoxin Systems in Eukaryotic Cells: Strategies and Applications. *Toxins*, *8*(2), 49. https://doi.org/10.3390/toxins8020049
12. Piscotta, F. J., Jeffrey, P. D., & Link, A. J. (2019). ParST is a widespread toxin-antitoxin module that targets nucleotide metabolism. *Proceedings of the National Academy of Sciences of the United States of America*, *116*(3), 826–834. https://doi.org/10.1073/pnas.1814633116
13. Wu, A. Y., Kamruzzaman, M., & Iredell, J. R. (2020). Specialised functions of two common plasmid mediated toxin-antitoxin systems, ccdAB and pemIK, in Enterobacteriaceae. *PloS one*, *15*(6), e0230652. https://doi.org/10.1371/journal.pone.0230652
14. Chan, C. T., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2016). 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. *Nature chemical biology*, *12*(2), 82–86. https://doi.org/10.1038/nchembio.1979
15. Amitai S, Yassin Y, Engelberg-Kulka H. MazF-mediated cell death in Escherichia coli: a point of no return. J Bacteriol. 2004 Dec;186(24):8295-300. doi: 10.1128/JB.186.24.8295-8300.2004. PMID: 15576778; PMCID: PMC532418.

#### Find Out More

* [Description](https://2021.igem.org/Team%3ALeiden/Description)
* [Proof of Concept](https://2021.igem.org/Team%3ALeiden/Proof_Of_Concept)
* [Implementation](https://2021.igem.org/Team%3ALeiden/Implementation)
* [Entrepreneurship](https://2021.igem.org/Team%3ALeiden/Entrepreneurship)
* [Integrated Human Practices](https://2021.igem.org/Team%3ALeiden/Human_Practices)

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# Team:Leiden/Experiments

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DOPLLOCK iGEM Leiden

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# Experiments

For building up DOPL LOCK, we designed and set up necessary experiments to demonstrate the proof-of-concept. In this page, you can find an overview of the experiment design and a summary of the experiments we performed.

Experiments

Protocols

# Experimental design

Because the toxin:antitoxin ratio to maintain the balance state was hard to find in previous literature, we used a **bottom-up approach** to build up the DOPL LOCK system. We divided the experiments into three stages. The first stage of the experiments was focusing on the **characterization** of each part in the DOPL LOCK system. For the characterization of the origin of replication (Oris), we co-transformed *Escherichia coli* (*E. coli*) TOP10 with two plasmids containing different fluorescent protein expression cassettes. Based on the fluorescent intensity of the strains, the best compatible pair of Oris was selected. For the characterization of toxins, we cloned inducible promoters in front of the toxin genes and performed a toxicity assay by measuring the bacterial growth after expression of the toxins was induced. For the promoters, we ligated promoters with mCherry to measure their transcriptional activity. For the second stage, we aim to create a single **toxin-antitoxin system** (TA system) based on the characterization from the first stage. The single TA system contains constructs of inducible promoter::toxin and constitutive promoter::antitoxin on a single plasmid. It will be used to find the balanced expression level. In the final stage, two verified single TA systems can be assembled and the final version of DOPL LOCK can be achieved.

![ Figure 1 overview of DOPL LOCK system](https://static.igem.org/mediawiki/2021/7/76/T--Leiden--Results-Figure1.png)

# Experiment 1: sfGFP-RFP swap

We swapped the sfGFP reporter cassette in the original JUMP plasmids (comprised of constitutive promoter [BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100) and sfGFP gene) to a RFP reporter cassette ([BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450)). This experiment created a second collection of JUMP plasmids with different reporters, which enabled us to evaluate the behavior of the co-transformed plasmids. The gene swap was done based on restriction enzyme cloning. JUMP plasmids and pSB1A3-RFP were both restricted by PstI and EcoRI, providing us with the JUMP backbone and an RFP construct. Then, the restriction products were mixed with a backbones/constructs molar ratio of 1:3. The DNA ligase and the buffer were added to the mixture for the reaction. Finally the reactions were stopped by heat shock and the products were ready for the transformations.

# Experiment 2: (Co-)transformation of sfGFP and RFP plasmids

![Figure 2 Selection of Oris](https://static.igem.org/mediawiki/2021/2/2e/T--Leiden--Results-Figure2.png)

*Figure 2: Selection of Oris*

*Table 1: The plasmids and their constructs used in the co-transformation*

| NAME | RESISTANCE | ORIGIN OF REPLICATION | INSERT | INCOMPATIBILITY GROUP | COPY NUMBER |
| --- | --- | --- | --- | --- | --- |
| pJUMP26 | Kanamycin | p15A | sfGFP | B | Medium/Low copy number |
| pJUMP27 | Kanamycin | pSC101 | sfGFP | C | Low copy number |
| pJUMP28 | Kanamycin | pUC | sfGFP | A | High copy number |
| pJUMP29 | Kanamycin | pBR322/ROB | sfGFP | A | Medium/High Copy number |
| pJUMP46 | Spectinomycin | p15A | RFP | B | Medium/Low copy number |
| pJUMP47 | Spectinomycin | pSC101 | RFP | C | Low copy number |
| pJUMP48 | Spectinomycin | pUC | RFP | A | High copy number |
| pJUMP49 | Spectinomycin | pBR322/ROB | RFP | A | Medium/High Copy number |

Both single- and co-transformations of JUMP plasmids were performed during our labtime. Detailed information of plasmids used in this experiment is shown in Table 1. Co-transformations were performed to test the compatibility of the different Oris on the different JUMP plasmids and therefore the stably maintenance of the plasmid (Figure 2). The sfGFP and RFP containing JUMP plasmids were co-transformed in the *E.coli* TOP10 strain. Co-transformations were performed by heat-shocking the sfGFP and RFP plasmids into TOP10 competent cells according to the co-transformation protocol. Next, the transformants are grown on sterile LB agar medium containing spectinomycin and kanamycin as a selection method. After successful transformation in *E.coli*, fluorescent markers were expressed and measured by plate reader assay. As a result, it was shown that the Oris p15A and pBR322/ROB were most suitable to express similar levels of protein ([Results page](https://2021.igem.org/Team%3ALeiden/Results)).

Single-transformations were often used as a positive control during plate reader assays, containing only a sfGFP or RFP plasmid. TOP10 strain containing no plasmids was used as negative control. The same protocol was used for the co-transformation, but in this case, only one plasmid was added to the competent TOP10 cells.

# Experiment 3: Biobrick ligation

Standardized parts and coding sequences needed for the DOPL LOCK system, including toxins, antitoxins, promoters, GFP, and mCherry were obtained as gBlocks from our sponsoring company Integrated DNA Technologies (IDT). The toxin gBlocks are double stranded DNA fragments that were cloned into the iGEM plasmids pSB1A3 and pSB1C3 by restriction enzyme cloning. The ligation was performed according to the Ligation of gBlock DNA into plasmids protocol by first diluting and digesting the gBlock DNA with EcoRI and PstI. Then, the digested gBlocks were ligated into the linearized pSB1A3 or pSB1C3 plasmids by EcoRI and PstI. We transformed these plasmids into DH5α strain to multiply their amounts. Ligating the gBlocks into both plasmids enables us to assemble composite parts that require the following experiments rapidly.

# Experiment 4: 3A assembly

3A assembly was intensively used in our project to create constructs for the characterization of different parts. The assembly was performed according to the [protocol in iGEM registry](http://parts.igem.org/Assembly%3A3A_Assembly). The plasmid products of 3A assembly were transformed into *E. coli* and grown on plates with kanamycin or spectinomycin. Only the strain containing the correct construct could survive. They were transferred to liquid LB medium to be prepared for the measurement of the optical density or the fluorescent intensity.

We cloned a variety of promoters (the Anderson collection of constitutive promoters and inducible promoters) in front of the fluorescent protein/toxin/antitoxin genes. For the promoter characterization, we assembled plasmids of pJUMP49-pBAD::mCherry and pJUMP49-p2547::mCherry. To evaluate the TA system, we assembled pJUMP26-pBAD::ccdB, pJUMP26-pBAD::HOK, pJUMP26-pBAD::RelE and pJUMP26-pBAD::MazF.

*For more information about these constructs, see  [Parts](https://2021.igem.org/Team%3ALeiden/Parts).*

# Experiment 5: Plate reader fluorescence assay

![Figure 3 Selection of the promoters](https://static.igem.org/mediawiki/2021/3/3b/T--Leiden--Results-Figure4.png)
*Figure 3: Selection of the promoters*

We calibrated the inducible promoter pBAD ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)) with the constitutive promoter [BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100) (referred to as p2547 in the following) using plate reader measurements (TECAN spark) (Figure 3). By doing this, we can relate these two kinds of promoters and use them to regulate toxin-antitoxin gene expressions. The plasmids used in the calibration were pJUMP49-pBAD::mCherry, pJUMP49-p2547::mCherry, and pJUMP49-pBAD (see [Parts page](https://2021.igem.org/Team%3ALeiden/Parts)). These plasmids were created by 3A assembly and transformed to the *E. coli* TOP10 strain. A 96-well plate with a gradient of L-arabinose was prepared and strains containing correct constructs were added to the plates in triplicates (more details in [Measurement page](https://2021.igem.org/Team%3ALeiden/Measurement)). After that, the 96-well plates were put in the plate reader. The temperature was maintained at 37 °C in the plate reader and the plate shaken at 120 rpm. Every 12 min the fluorescence and the optical density of each plate were measured for 10 h.

The plate reader was also used to evaluate the compatibility of Oris when co-transformed. The co-transformed cells were cultured overnight in LB-medium and pippeted to a 96-well plate in triplicates accordingly. Each overnight culture was continually diluted 5 times to another three wells. Then, the plate was put in the plate reader for a one-time measurement of fluorescence and optical density. The temperature and the shaking speed were the same as previously mentioned.

# Experiment 6: Toxicity assay in liquid medium

For the evaluation of the toxicity of toxins, we first ligated the inducible promoter pBAD with each toxin in the pJUMP26 backbone (see [Parts page](https://2021.igem.org/Team%3ALeiden/Parts)). In this way, the toxin gene expression was regulated by the L-arabinose concentration in the environment. After successful transformation of the desired constructs in *E. coli* TOP10, the strains were inoculated in LB-medium overnight. Then, they were diluted 5 times and pipetted to a 96-well plate in triplicates. Different amounts of L-arabinose were added accordingly to each well. Ampicillin was added to some wells to achieve full cell death as positive controls. Strains containing pBAD-mCherry were used as a negative control to exclude the inhibitory effect of overexpression. The optical density of these cells was measured by plate readers continuously for 10 h to evaluate their viability after toxin expression was induced. The temperature was maintained at 37 °C in the plate reader and the plate shaken at the speed of 120 rpm. Every 12 min the fluorescence and the optical density of each plate were measured.

We also applied minimal medium instead of LB-medium for the toxicity assay. The procedures were similar as described above, except for using M9 minimal medium to prepare the 96-well plate.

# Experiment 7: Toxicity assay by LB-agar plate

By plating cells with and without toxin expression, we can obtain different cell viability when toxin expression is induced. To do this, the agar plates with different amounts of L-arabinose as supplements were first prepared. Then, the strains containing different pBAD::toxin constructs were diluted until optical density reached 0.1. Then 5 μL of the bacterial solution were pipetted at the same position of the plates. The plates were put in the incubator overnight at 37 °C. The diameters of the colonies indicated cell viability with or without toxin induction.

# Experiment 8: PCR-based cloning

As the promoters we used were less than 100 bp, we noticed difficulty trying to clone the promoters into plasmids. Therefore, primers were developed to ligate the p162 promoter in front of the ccdB, ccdA, RelE and RelB by PCR-based cloning (the list of the primers are shown in Table 2). For the PCR reaction a PCR gradient was performed according to PCR-based cloning protocol. To verify if the promoter was successfully ligated in front of the toxins, gel electrophoresis was performed. We succeeded in creating these constructs via PCR cloning (see [Results page](https://2021.igem.org/Team%3ALeiden/Results)). Thereafter, the obtained constructs were used further trying to create the DOPL LOCK system. Although this was not successful due to time constraints, this can still be researched further in the future.

*Table 2: The primers used in PCR-based cloning*

| RelE\_Forward | ATTATGAATTCTAGAGGATCCCTGATGGCTAGCTCAGTCCTAGGGATTATGCTAGCAAAGAGGAGAAATACTAGGTGAGCGAC |
| --- | --- |
| RelB\_Forward | ATATTGAATTCTAGAGGATCCTTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAGCAAAGAGGAGAAATACTAGATGGGTAGCATTAACC |
| ccdB\_Forward | ATTATGAATTCTAGAGGATCCCTGATGGCTAGCTCAGTCCTAGGGATTATGCTAGCAAAGAGGAGAAATACTAGATGCAGTTTAAGGTTTACA |
| ccdA\_Forward | ATATTGAATTCTAGAGGATCCTTGACAGCTAGCTCAGTCCTAGGGATTGTGCTAGCAAAGAGGAGAAATACTAGATGAAGCAGCG |
| suffix antitox\_Reverse | CCTGCAGCGGCCGCTACTAGTA |
| suffix \_Toxin\_Reverse | CTGCAGCGGCCGCTACTAGTA |

# Experiment 9: Competent cells preparation

Two different *E.coli* strains were used during our lab time, including DH5α and TOP10. DH5α was first used to test the overexpression of toxins, although the DH5α strain metabolized the arabinose. As a result, the promoters were not induced enough to cause bacterial death. The TOP10 bacterial strain was then used for the plate reader assay to induce the toxins. The protocol *E.coli* competent cells TSS method was used to make competent cells of both *E. coli* strains. First, the cells were inoculated in liquid LB medium until the culture reached an OD600 between 0.3-0.4.

After centrifugation 5 mL of cold TSS medium was added to the cells and cooled down in liquid nitrogen. Ultimately, the competent cells were stored at -80 °C and thawed before use.

[x]

* # Ligation of gBlock DNA into plasmids

  gBlock DNA of toxin, antitoxin, and promoters was obtained from IDT.

  ## Consumables

  + pSB1A3 or pSB1C3 linearized with EcoRI-HF and PstI
  + gBlock DNA, IDT
  + EcoRI-HF
  + PstI
  + T4-Ligase
  + T4-ligase buffer

  ## Equipment

  + Incubator at 37°C
  + Vortex
  + Centrifuge

  ## Resuspension of gBlock DNA

  Centrifuge the tube for 5 sec at a minimum of 3000 x g to pellet the material to the bottom of the tube.

  Add 20 ul AE-buffer to the IDT DNA.

  Briefly vortex and centrifuge

  Store in -20

  ## Digestion of gBlock DNA

  *Table 1: Master mix schedule*

  | Components | 1 reaction (µL) | Master mix for X reactions (µL) |
  | --- | --- | --- |
  | gBlock DNA (10 ng/µL) | 7 | N/A |
  | EcoRI-HF | 1 | (X + 1) \* 1 |
  | PstI | 1 | (X + 1) \* 1 |
  | 10x Cutsmart buffer | 1 | (X + 1) \* 1 |
  | MiliQ | 0 |  |
  | Total volume | 10 | (X + 1) \* 10 |

  Transfer 10µL of the gblock DNA from the Stock tube to a fresh 1.5mL tube.

  Add 20µL of the master mix to the gBlock DNA

  Vortex briefly and spin down at 3000 RPM for 1 second

  Incubate in 37°C for 1-2 hours

  Heat inactivate at 80 °C for 20 minutes.

  Use immediately or store in -20°C

  ## Ligation of Biobrick in Plasmid

  Mix the following components in a 1.5 mL tube, for ratios between insert and vector, check table 2, calculated via: https://nebiocalculator.neb.com/#!/ligation, a ratio of 1:5 was used.

  Incubate at RT for 2h or overnight.

  Transform in *E. coli* and pick the white colonies.

  *Table 2: Ligation mixture*

  | Components | 1 reaction (µL) |  |
  | --- | --- | --- |
  | Linearized plasmid (20ng/µL) | 2 | 30 |
  | Digested gBlock DNA (3.33 ng/µL) | 10 |  |
  | T4-ligase | 1 | 15 |
  | 10x T4-ligase buffer | 2 | 30 |
  | MiliQ | 4 | 60 |
  | Total volume | 20 |  |
  |  |  |  |

  ## *Table 3: Overview of ligation in Biobrick plasmids*

  | Vector | Insert |  |  |  |  |
  | --- | --- | --- | --- | --- | --- |
  | Name | Size (KB) | Amount (ng/reaction) | Name | Size (KB) | Amount (ng/reaction) |
  | pSB1C3 | 2170 | 20 | p1 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p21 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p162 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p387 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p623 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p908 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p1303 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p1487 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p1831 | 125 | 5.8 |
  | pSB1C3 | 2170 | 20 | p2547 | 125 | 5.8 |
  | pSB1A3 | 2155 | 20 | p\_pBAD\_CcdB\_T\_S | 1700 | 78.9 |
  | pSB1A3 | 2155 | 20 | p\_pLacI\_CcdA\_T\_S | 1673 | 77.6 |
  | pSB1A3 | 2155 | 20 | CcdB T | 482 | 22.4 |
  | pSB1A3 | 2155 | 20 | CcdA AT | 395 | 18.3 |
  | pSB1A3 | 2155 | 20 | MazF T | 512 | 23.8 |
  | pSB1A3 | 2155 | 20 | MazE AT | 425 | 19.7 |
  | pSB1A3 | 2155 | 20 | RelE T | 470 | 21.8 |
  | pSB1A3 | 2155 | 20 | RelB AT | 416 | 19.3 |
  | pSB1A3 | 2155 | 20 | HOK T | 603 | 28.0 |
  | pSB1A3 | 2155 | 20 | SOK AT | 389 | 18.1 |
  | pSB1A3 | 2155 | 20 | pBAD | 1263 | 58.6 |
  | pSB1A3 | 2155 | 20 | pLacI | 1323 | 61.4 |
  | pSB1A3 | 2155 | 20 | pTetOn | 581 | 27.0 |
  | pSB1A3 | 2155 | 20 | LacI RFP cassette | 1122 | 52.1 |
  | pSB1A3 | 2155 | 20 | GFP | 896 | 41.6 |
  | pSB1A3 | 2155 | 20 | mCherry | 890 | 41.3 |
  | pSB1A3 | 2155 | 20 | lambda t0 terminator | 148 | 6.9 |

[x]

* # OD to CFU calibration

  The aim is to relate optical density measured by spectrophotometer or plate reader to colony counting units. So we can estimate the cell count directly from the OD600 value.

  ## OD test

  -
   *E. coli*  DH5α
  - Petri dishes
  - Eppendorf 2mL
  - Eppendorf 1mL
  - Falcon tube 15mL

  1. Measure the OD600 of your cell cultures, making sure to dilute to the linear detection range of your plate reader.
  2. Dilute your overnight culture to OD600 gradient in 1mL of LB + antibiotics media. Do this in triplicate for each culture.
  3. Use (C1)(V1) = (C2)(V2) to calculate your dilutions (C1 is your starting OD600; C2 is your target OD600 (= 0.1); V1 is the unknown volume in μL; V2 is the final volume (= 1000 μL)
  4. Dilute the OD600 to a certain value (Shown in the 96-well plate).
  5. Recommended plate setup is below. Each well should have 200 μL. Put the plate in the plate reader. And test their OD600 via spectrophotometer immediately.

  *Table 4: Layout well*

  | OD0.05A | OD0.05B | OD0.05C | OD0.1A | OD0.1B | OD0.1C |  |  |  |  |  |  |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | OD0.2A | OD0.2B | OD0.2C | OD0.4A | OD0.4B | OD0.4C |  |  |  |  |  |  |
  | OD0.8A | OD0.8B | OD0.8C |  |  |  |  |  |  |  |  |  |
  | Blank control A | Blank control B | Blank control C |  |  |  |  |  |  |  |  |  |
  |  |  |  |  |  |  |  |  |  |  |  |  |
  |  |  |  |  |  |  |  |  |  |  |  |  |
  |  |  |  |  |  |  |  |  |  |  |  |  |
  |  |  |  |  |  |  |  |  |  |  |  |  |

  1. Do the following serial dilutions for your triplicate Starting Samples you prepared in Step 3. You should have 18 total Starting Samples. For OD=0.05, dilute 1:20, 1:20, 1:10, 1:10, 1:10; For OD=0.1, dilute 1:20, 1:20, 1:20, 1:10, 1:10; For OD=0.2, dilute 1:40, 1:20, 1:20, 1:10, 1:10; For OD=0.4, dilute 1:40, 1:40, 1:20, 1:10, 1:10; For OD=0.8, dilute 1:8, 1:20, 1:20, 1:10, 1:10, 1:10
  2. You will need 3 Liters of LB Agar (45 petri dishes in total)
  3. Label each tube according to the figure above (Dilution 1, etc.) for each Starting Sample
  4. Split the LB plate in three parts and spead 33.3 μL of the sample on each part of the plate.
  5. Incubate at 37 °C overnight and count colonies after 18-20 hours of growth
  6. Count the colonies on each plate with fewer than 300 colonies
  7. Next, multiply the colony count by the Final Dilution Factor on each plate

[x]

* # Inducible promoters

  Experiments for the model and to test the amount of protein produced by the inducible promoters.

  ## Testing the inducible promoters

  1. Spectrophotometer
  2. Plate-reader
  3. Cells with plasmids
     1. GFP + LacI promoter
     2. GFP + pBAD promoter
     3. GFP + TetON promoter
     4. mCherry + LacI promoter
     5. mCherry + pBAD promoter
     6. mCherry + TetON promoter
  4. Inducer
  5. Media
  6. Cell counter

  ## Calibration

  Make a calibration curve between OD600 (0 - 1.5) and number of cells by measuring the OD & cell count of different values.

  Make a reference for the fluorescence and amount of GFP protein.

  ## Preparation

  Transform the cells with the plasmids

  Dilute the cells to about 0.1 and add them in the 96-well plate

  Grow the cells up to OD600 = 0.5

  Add certain concentration inducer (according to the table below)

  ## Measuring Fluorescence

  Measure the fluorescence and OD600 every 10 minutes during 5 hours with the plate reader

  *Table 5: Well layout*

  | C1 | C1 | C1 | B1 | B1 | B1 | L1 | L1 | L1 | T1 | T1 | T1 |
  | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
  | C2 | C2 | C2 | B2 | B2 | B2 | L2 | L2 | L2 | T2 | T2 | T2 |
  | C3 | C3 | C3 | B3 | B3 | B3 | L3 | L3 | L3 | T3 | T3 | T3 |
  | C4 | C4 | C4 | B4 | B4 | B4 | L4 | L4 | L4 | T4 | T4 | T4 |
  | C5 | C5 | C5 | B5 | B5 | B5 | L5 | L5 | L5 | T5 | T5 | T5 |
  | C6 | C6 | C6 | B6 | B6 | B6 | L6 | L6 | L6 | T6 | T6 | T6 |
  | C7 | C8 | C9 | B7 | B7 | B7 | L7 | L7 | L7 | T7 | T7 | T7 |
  |  |  |  | B8 | B8 | B8 | L8 | L8 | L8 | T8 | T8 | T8 |

  *Table 6: Overview*

  | C1 | Media with antibiotics |
  | --- | --- |
  | C2 | Media with antibiotics with E. coli without plasmid |
  | C3 | Media with antibiotics with E. coli with pBAD |
  | C4 | Media with antibiotics with E. coli with LacI |
  | C5 | Media with antibiotics with E. coli with TetON |
  | C6 | Media with antibiotics with E. coli with plasmid |
  | C7 | E. coli with pBAD - 0.0 mM |
  | C8 | E. coli with lacI - 0.0 mM |
  | C9 | E. coli with TetON - 0.0 Mm |
  |  |  |
  | B1 | E. coli with pBAD - 0.01 mM |
  | B2 | E. coli with pBAD - 0.02 mM |
  | B3 | E. coli with pBAD - 0.05 mM |
  | B4 | E. coli with pBAD - 0.1 mM |
  | B5 | E. coli with pBAD - 0.2 mM |
  | B6 | E. coli with pBAD - 0.5 mM |
  | B7 | E. coli with pBAD - 0.8 mM |
  | B8 | E. coli with pBAD - 1 mM |
  |  |  |
  | L1 | E. coli with lacI - 0.01 mM |
  | L2 | E. coli with lacI - 0.02 mM |
  | L3 | E. coli with lacI - 0.05 mM |
  | L4 | E. coli with lacI - 0.1 mM |
  | L5 | E. coli with lacI - 0.2 mM |
  | L6 | E. coli with lacI - 0.5 mM |
  | L7 | E. coli with lacI - 0.8 mM |
  | L8 | E. coli with lacI - 1 mM |
  |  |  |
  | T1 | E. coli with TetON - 0.01 mM |
  | T2 | E. coli with TetON - 0.02 mM |
  | T3 | E. coli with TetON - 0.05 mM |
  | T4 | E. coli with TetON - 0.1 mM |
  | T5 | E. coli with TetON - 0.2 mM |
  | T6 | E. coli with TetON - 0.5 mM |
  | T7 | E. coli with TetON - 0.8 mM |
  | T8 | E. coli with TetON - 1 mM |

[x]

* # FACS preparation

  ### Prepare FACS sample

  1. [FACS tubes](https://www.pluriselect.com/nl/catalog/product/view/id/450#size=29) + filter/strainer (40 um)
  2. Cell culture
  3. PBS
     1. NaCl: 137 mM
     2. KCl: 2.7 mM
     3. Na2HPO4: 10 mM
     4. KH2PO4: 1.8 mM
     5. All in MiliQ water

  Spin-off 1 mL of cell suspension (OD between 0.1-0.5 but higher is also okay)

  Take away supernatant

  Resuspend in 1 mL PBS

  Drop onto 40 um filter (presoaked in PBS)

  200 uL of the filtered liquid can be put through the FACS machine

  ## Running the FACS

  Startup the machine (before sample prep)

  When the machine is warmed up, select protocol

  Screenshot settings

  Run blank to find background

  Put 200 uL sample in cell sorter tubes

[x]

* # 3A Assembly

  Assembly promoter and insert gene into new plasmid backbone

  ## Calculation

  calculate the amount of plasmid and MilliQ needed for each digestion using the [NEB calculator](https://nebiocalculator.neb.com/#!/ligation) for your desired molar ratios. The molar ratio needed depends on the length of the inserts and backbone.

  ## Digestion

  Find corresponding restriction enzymes for each plasmid.

  Plasmid backbone: EcoRI, PstI

  Promoter: EcoRI, SpeI

  Insert gene: Xbal, PstI

  ![](https://static.igem.org/mediawiki/parts/4/42/3AAssembly.png)

  The ligation solution should contain the following parts (uL):

  Plasmid concentration need to be measured in advanced

  Plasmid should be added at last

  Enzymes and smartbuffer should always be put on ice and stored immediately after it once has been used

  *Table 7: Digestion mixture*

  | Enzyme 1 | 0.5 |  | Plasmid ID | Plasmid concentration | Plasmid | water |
  | --- | --- | --- | --- | --- | --- | --- |
  | Enzyme 2 | 0.5 |  |  | 0 | x | x |
  | buffer | 1 |  |  | 0 | x | x |
  | MiniQ | 8-X |  |  | 0 | x | x |
  | Plamsid | X | 100ng in total |  | 49 | 2.0408163265 | 5.9591836735 |

  Incubate the digestion solution at 37 degrees for 1h

  Heat shock 80 °C for 20min to stop the enzymatic reaction

  ## Ligation

  The ligation solution should contain the following parts (uL): see table 8

  The amount of gene should have equal molar weight to the plasmid

  Using the NEB calculator.

  *Table 8: Ligation mixture*

  | Plasmid back bone | 2 |  |
  | --- | --- | --- |
  | gene 1 | X | \<3 |
  | gene 2 | Y | \<3 |
  | T4 ligase | 0.5 |  |
  | T4 ligase buffer | 1 |  |
  | MiniQ | 6.5-X-Y |  |

  Leave the solution at room temperature and react for 1.5h

  Heatshock at 80 °C for 20 min

  Use 5uL (half) of the ligation solution to transform the bacteria.

[x]

* # Co-transformation

  Prepare dilutions of the plasmids with a concentration of 2 ng/µL

  Switch on the water bath and set temperature at 42 °C. Also, turn on the heat/shaking-block and set up to 37 °C

  Load a bucket with ice from the ice machine

  Take the bacterial cells and SOC (Super optimal broth with catabolite repression) out of the -80 °C freezer. Transfer the cells directly to ice. Do not touch the bottom of the tube that contains the cells.

  Thaw the cells on ice for ~5 minutes

  Add 1 μL of each plasmid into 20 μL bacteria. Mix well. Make sure you work near the Bunsen burner flame

  Leave the cells on ice for 5 minutes

  Heat-shock the cells for 30 seconds (exactly!) at 42°C

  Return the cells directly to ice for 2 minutes

  Add 80 μL of SOC solution to the bacteria

  Incubate for 60 minutes at 37 °C and 300 rpm

  Dry agar plate, supplemented with Spectinomycin(25 µg/mL) and Kanamycin(30 µg/mL) in the 37 °C incubators. Place the plate upside down and slightly opened.

  ## Plating the cells on agar plate

  Take the dried agar plate out of the 37 °C incubators

  Label the bottom of the plates

  Open an agar plate in close proximity of the Bunsen burner flame

  Pipette the cells (100 μL) on the plate

  Sterilize the Drigalski spatula by burning the alcohol on it, shortly let it cool down

  Spread the cells on the plate using the sterile spatula

  Transfer the agar plate to the 37°C incubator

  Place the plate upside down, closed • Let the cells grow on the plate overnight.

[x]

* # E. coli transformation

  This protocol describes how to transform  [competent *E. coli.\_\_Protocol*](https://2018.igem.org/Team%3ALeiden/Experiments)  is adapted from Marjolein Crooijmans, iGEM 2018

  1. Competent *E.coli* cells,
  2. Plasmid DNA or Ligation reaction
  3. Ice
  4. Waterbath at 42°C
  5. LB medium
  6. LB plates with appropriate antibiotics
     1. Ampicillin (100 µg/mL)
     2. Kanamycin: (50 µg/mL)
     3. Chlorampenicol: (25 µg/mL)
     4. Spectinomycin: (50 µg/mL)

  Thaw 50 ul competent cells on ice

  Add 10 ul ligation mix DNA and mix carefully

  30-60 min on ice (30 min is completely fine)

  Incubate 90s 42oC (heat-shock) and immediately place back on ice

  Add 1 ml LB medium (do 400 uL instead)

  Incubate 1.5 hour at 37OC without shaking (1 hour is also fine)

  Plate on LB agar plates with appropriate antibiotics

  Incubate overnight at 37OC

[x]

* # PCR-based cloning

  Ligating promoters in front of the toxins with PCR-based protocol.

  PCR reaction

  DNTPs

  Forward primer

  Reverse primer

  Taq polymerase

  Taq buffer

  *Table 9: PCR mixture*

  | DNA | 1 ul |
  | --- | --- |
  | Promoter | 1 ul |
  | Forward primer | 2 ul |
  | Reverse primer | 2 ul |
  | Taq DNA buffer | 2 ul |
  | Taq polymerase | 0.25 ul |
  | MilliQ | 16.75 ul |
  | Total | 25 ul |

  *Table 10. PCR gradient program*

  | Phase | Repetitions | Temperature (C) | Time (s) | Notes |
  | --- | --- | --- | --- | --- |
  | 1 | 1 time | 95 | 30 |  |
  | 2 | 15 times | 95 | 30 |  |
  | 2 |  | 68 | 30 | -0.6 C / cycle |
  | 2 |  | 72 | 60 |  |
  | 3 | 20 times | 95 | 30 |  |
  | 3 |  | 59 | 30 |  |
  | 3 |  | 72 | 60 |  |
  | 4 | 1 time | 72 | 300 |  |
  | 4 | 1 time | 4 | infinity |  |

[x]

* # Competent cell CFU test

  1. Shaker
  2. Consumable
     1. Eppendorf tube \*9
     2. LB medium
     3. Ice
  3. Competent cells
  4. Plasmid DNA

  Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tubes for each transformation and then pre-chill by placing the tubes on ice.

  Do triplicates (3 each) of each concentration if possible, so you can calculate an average colony yield.

  Spin down the DNA tubes containing your controls to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient.

  Pipet DNA into each microcentrifuge tube. Recommend final concentration in 1mL medium is 0.01ng/mL, 0.05ng/mL and 0.1ng/mL

  Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.

  Incubate on ice for 30 minutes.

  Pre-heat waterbath now to 42°C. Otherwise, hot water and an accurate thermometer works, too!

  Heat-shock the cells by placing into the waterbath for 45 seconds (no longer than 1 min). Be careful to keep the lids of the tubes above the water level, and keep the ice close by.

  Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes.

  Add 950 µL of SOC media per tube, and incubate at 37°C for 1 hour shaking at 200-300rpm.

  Prepare the agar plates during this time: label them, and add sterile glass beads if using beads to spread the mixture.

  Pipet 100 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.

  Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation.

  Efficiency (in cfu/µg) = [colonies on plate (cfu) / Amount of DNA plated (ng)] x 1000 (ng/µg)

  *Note: The measurement "Amount of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:*

  Amount of DNA plated (ng) = Volume DNA added (1 µL) x concentration of DNA (refer to vial, convert to ng/µL) x [volume plated (100 µL) / total reaction volume (1000 µL)]

[x]

* # Electroporation protocol

  This method of competent cell preparation follows the TSS preparation described by Chung & Miller. Chung, C. T., & Miller, R. H. (1993). Preparation and storage of competent Escherichia coli cells. *Methods in enzymology*, *218*, 621–627. https://doi.org/10.1016/0076-6879(93)18045-e

  1. Bacteria
     1. *E. coli* strains of choice, for example DH5α
  2. Consumables
     1. dimethyl sulfoxide (DMSO)
     2. polyethylene glycol (PEG)
     3. Ice
     4. Liquid nitrogen
     5. 2 M MgCl2 (1.94 g in 100mL)
     6. LB medium
  3. Equipment
     1. 250 ml Erlenmeyer
     2. Centrifuge
     3. Shaking incubator
     4. Spectrophotometer
     5. 50 mL tubes

  *Table 11. TSS medium (pH 6.5)*

  | Component | Stock (M) | Amount | Unit | Final concentration |  |
  | --- | --- | --- | --- | --- | --- |
  | MgCl2 | 1 | 2.5 | ml | 50 | mM |
  | DMSO | - | 2.5 | ml | 5 | % |
  | PEG -6000 | - | 5 | g | 10 | % |
  | LB |  | up to 42.5 | ml | - | - |

  ## Day 1

  Grow preculture LB (10 ml) at 37OC overnight

  Prewarm 500 ml medium at 37OC

  ## Day 2

  Inoculate 2x 2.5 ml preculture in 250 ml prewarmed LB medium

  Grow 2-2.5 hours at 37OC to OD600 = 0.3-0.4 (not \>0.4)

  Pellet cells by centrifugation (10min, 4000rpm centrifuge 5 degrees)

  Resuspend pellet in 5 ml cold (!) TSS medium

  Divide suspension in 50 ul portions

  Immediately cool down each tube in liquid nitrogen

  Store at -80 °C

  Note: This protocol does not require the administration of a heat shock: incubating the cells with the DNA on ice is enough to obtain high transformation rates. According to the article, administering a heat shock may lower transformation efficiencies.

[x]

* # E. coli competent cells TSS method

  ### Preparation of LB agar plates

  1. Consumables
     1. LB medium 25 g/L
     2. Bacterial Agar 15g/L
  2. Antibiotic stocks (1000X)
     1. Ampicillin (100 mg/mL)
     2. Kanamycin: (50 mg/mL)
     3. Chlorampenicol: (25 mg/mL)
     4. Spectinomycin: (50 mg/mL)
  3. Equipment
     1. Autoclave
     2. Bunsen burner
     3. Incubator

  Microwave solidified LB agar bottles until they are almost boiling or use freshly autoclaved LB agar.

  Let the agar cool until you can hold it in your hands for 10 seconds

  **Optional:** For selection plates add for 1 mlof LB agar,1 µL of the antibiotic stock

  Pour 25 mL LB agar in a petri-dish and let the agar solidify.

  Label the Petri dishes with the appropriate color corresponding to the antibiotics used!

  Store the plates upside down at 5°C

[x]

* # Standard protocol for pouring plates

  Making your own [electrocompetent cells](https://international.neb.com/protocols/2012/06/21/making-your-own-electrocompetent-cells)

  ## Media

  **SOB**

  1. 2% tryptone
  2. 5% yeast extract
  3. 10 mM NaCl
  4. 5 mM KCl
  5. 10 mM MgCl2
  6. 10 mM MgSO4

  **SOC**

  - SOB + 20 mM glucose

  Appropriate Antibiotics for Your Application.

  Antibiotics for Plasmid selection.

  Antibiotic Working Concentration

  1. Ampicillin 100 µg/ml
  2. Carbenicillin 100 µg/ml
  3. Chloramphenicol 33 µg/ml
  4. Kanamycin 30 µg/ml
  5. Streptomycin 25 µg/ml
  6. Tetracycline 15 µg/ml

  Sterile 10% glycerol (can be autoclaved) is needed for the washes. The volume of 10% glycerol needed is 2X the culture volume (for example, a 500 ml culture requires 1L of 10% glycerol). Procedure (for 2, 250 ml cultures)

  1. Inoculate 1 colony from a fresh plate of the strain to be made electrocompetent into 10 ml of SOB in a 125 ml flask and incubate for 16-18 hours at 37°C and 250 rpm.
  2. Have ready 2, 1 L flasks containing 250 ml each of SOB pre-warmed to 37°C. Add two drops of the overnight culture to each of the flasks. Shake at 37°C and 250 rpm until the cultures reached an OD600 of 0.5-0.7. Be sure to turn on the centrifuge and cool rotor to 4°C well in advance of harvesting cells. Be sure to place 1 L of 10% glycerol on ice well in advance of harvesting cells
  3. Place cultures on ice for 15 minutes. From this point on the cultures must be kept ice cold. Pour each 250 ml culture into chilled 500 ml (or 1000 ml) centrifuge bottles.
  4. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant and aspirate any residual broth. Add 250 ml of glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting up and down.
  5. Centrifuge at 5000 rpm for 10 min. Pour off the supernatant, it is not necessary to aspirate. Completely suspend the cells in 250 ml glycerol and re-centrifuge.
  6. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down. If necessary, adjust the final volume of cells so that the OD600 is within the range 210-270.
  7. At this point you can electroporate or freeze the cells away. To freeze, add 100 microliters of the culture to microcentrifuge tubes on ice. Once you have used all of the cultures, transfer the tubes to dry ice for 10 minutes. Once the cultures are frozen, transfer them to a -80 °C freezer. The cultures should be good for \>6 months.

  ## Electroporation Protocol

  1. The electroporation protocol will vary depending on the strain so this protocol may need to be optimized.
  2. For control electroporation dilute pUC19 to 10 pg/µl with Milli-Q water.
  3. Turn on electroporator and set to 1.7-2.5 kV (optimize for strain), 200 ohms and 25 µF. Place recovery SOC in 37°C water bath. Pre-warm LB-antibiotic plates at 37°C. Thaw cells on ice for 10 min or use freshly made cells. Place appropriate number of microcentrifuge tubes and 1 mm-electroporation cuvettes on ice .
  4. Flick the tube containing cells a few times to mix and add 25 µl to the microcentrifuge tubes.
  5. Add 1 µl of a 10 pg/µl DNA solution (in DI water) to the cells in the microcentrifuge tube.
  6. Transfer the DNA-cell mixture to the cold cuvette, tap on countertop 2X, wipe water from exterior of cuvette and place in the electroporation module and press pulse (don't hold the button down).
  7. Immediately add 975 µl of 37°C SOC, mix by pipetting up and down once and transfer to a 15 ml-falcon tube.
  8. Rotate in the 37°C incubator for 1 h.
  9. Make appropriate dilutions. When using 10 pg of DNA, make two dilutions: Dilute 10 µl cells into 990 µl SOC and plate 100 µl. (1000-fold dilution) Dilute 100 µl cells into 900 µl SOC and plate 100 µl. (100-fold dilution) Incubate overnight at 37°C. Calculation: If the culture was diluted 1000-fold when plated, the total cfu per ml is 1000 times the number of colonies counted. The cfu is divided by the amount of pUC19 (10 pg per ml) cfu/ µg = (colonies counted\*1000) / (0.00001 µg pUC19)

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# Team:Leiden/Results

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DOPLLOCK iGEM Leiden

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# Results

After we applied the **bottom-up** design to build up DOPL LOCK, we performed experiments to **characterize components** of our system, including origin of replications, promoters, toxins and antitoxin. Then, we focused on **assembling the most compatible components** to the DOPL LOCK system. On this page, **detailed experimental results** that led to the [proof-of-concept](https://2021.igem.org/Team%3ALeiden/Proof_Of_Concept) are shown.

Overview

Results

Discussion

* [Introduction](#Introduction)
* [Characterization of parts](#Characterization_of_parts)
* [Part assembly](#Part_assembly)
* [Discussion](#Discussion)

# Introduction

**Preventing horizontal gene transfer** (HGT) and physically constraining GMOs are the most challenging issues to **broaden** the application of synthetic biology outside the labs. We designed DOPL LOCK to both **minimize** the chance of HGT and prevent the physical spread of the GMOs. In our system, the toxin-antitoxin (TA) expression needs to be balanced at a certain level. We applied the **bottom-up design** to build up DOPL LOCK in three steps. First, we started with the **characterization** of each genetic part that is influential to this balance, namely the origin of replication (Ori) of the plasmid vector, promoters upstream of the toxin and antitoxin genes, and the toxin-antitoxin genes themselves. All parts characterized in this project are shown in **Figure 1**. Secondly, we chose the **best compatible parts** and **assembled** them into a **single TA system** to find the balanced expression level. Finally, two verified TA systems can be assembled into the final version of DOPL LOCK on two different plasmids. During our lab time, we successfully characterized each part and found the **best combinations** of genetic parts, with the exception of the antitoxins. However, we failed to assemble a TA system in a single plasmid due to limited laboratory time. The results of our experiments are summarized as follows.

![Figure 1 overview of DOPL LOCK system](https://static.igem.org/mediawiki/2021/7/76/T--Leiden--Results-Figure1.png)

In the following part, we summarized the results of our experiment. More detailed information is shown in the Results section.

# Characterization of parts

## Origin of replication

> **Aim: Determine which combination of Oris maintained plasmid copy number in the cells at similar levels.**
>
> ✔ Successfully construct pJUMP plasmid collection with the RFP cassette as reporter.
>
> ✔ Single transformation showed that the plasmid copy number had minor influence on the fluorescent protein production after overnight incubation.
>
> ✔ p15A and pBR322/ROB exhibited the best compatibility.
>
> ✔ pSC101 and pUC have low expression in RFP when co-transformed.
>
> ❔ Substitute mRFP1 with mCherry to resolve FRET with sfGFP.
>
> (✔ and ❔ show "successful" or "remains a challenge")

Oris significantly influenced the plasmid copy number (Figure 2) [1]. To determine which Oris pair can maintain the plasmid copy number in the cell at similar levels, we used the JUMP plasmid collection that includes four types of Oris, two antibiotics selection markers and sfGFP reporter cassettes [2]. The sfGFP cassettes of the plasmids were switched to a RFP cassette () accordingly (all constructs are shown in the [Parts page](https://2021.igem.org/Team%3ALeiden/Parts)). Both vectors carrying sfGFP or RFP were co-transformed into the *E. coli* TOP10 strain and their fluorescence was measured by the plate reader.

![Figure 2 Selection of Oris](https://static.igem.org/mediawiki/2021/2/2e/T--Leiden--Results-Figure2.png)

*Figure 2: Selection of Oris*

The result showed that the Oris p15A and pBR322/ROB were the most compatible groups. Their co-transformed strain showed high levels of both sfGFP and RFP fluorescence signals (Figure 3). With this Ori combination, we could regulate the TA gene expression in two plasmids easily, since these two plasmid backbones have a more similar expression of the foreign genes they carried.

![Figure 3 Fluorescence of co-transformed p15A and pBR322/ROB (in the middle) with their single-transformed control group (at the sides) with error bars. Green bars represent the sfGFP fluorescent signal while red bars represent RFP signals.](https://static.igem.org/mediawiki/2021/1/11/T--Leiden--Results-Figure3.png)

*Figure 3: Fluorescence of co-transformed p15A and pBR322/ROB (in the middle) with their single-transformed control group (at the sides) with error bars. Green bars represent the sfGFP fluorescent signal while red bars represent RFP signals.*

We also designed experiments using the fluorescent protein pair sfGFP-mCherry to avoid Förster resonance energy transfer between sfGFP-RFP [2]. However, the cloning didn't succeed as there were no correct plasmid constructs in the gel.

## Promoters

> **Aim: Determine the arabinose inducibility of promoter pBAD and the relative transcription ability of pBAD** ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)) **to constitutive promoter p2547** ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)) **.**
>
> ✔ Successfully ligated the pBAD and p2547 promoter to mCherry ([BBa\_K3962340](http://parts.igem.org/Part%3ABBa_K3962340) [BBa\_K3962339](http://parts.igem.org/Part%3ABBa_K3962339)).
>
> ✔ Tested the inducibility of L-arabinose on pBAD
>
> ✔ Calibration of pBAD with constitutive promoter p2547.
>
> ❔ Calibration of other constitutive promoters in BBa\_J23100 promoter family.

The combination of a high-expression promoter of antitoxin and a relatively low-expression promoter of toxin is ideal for balancing a single TA system [4]. This approach prevents creating unnecessary constructs of constitutive promoters ligated with toxins. To simplify the experiment further, we first used mCherry as a reporter protein to characterize the inducibility of pBAD ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)). Afterwards, we compared the transcriptional activity of pBAD with constitutive promoter p2547 ([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)) (Figure 4).

![Figure 4 Selection of the promoters](https://static.igem.org/mediawiki/2021/3/3b/T--Leiden--Results-Figure4.png)

*Figure 4: Selection of the promoters*

The results showed pBAD was tightly regulated by L-arabinose from concentration 0.00032% to 1% (w/v). Then, under the same condition, we relate mCherry expression of pBAD to constant expression of constitutive promoter p2547 (Figure 5). Their correlation was fit into a linear regression with R2 = 0.9586. The characterization of this part provides us with crucial information to select promoters based on the predicted toxin/antitoxin ratio in equilibrium states. The selected promoters can be used to assemble a single TA system to balance their expression.

![Figure 5The fluorescence intensity of strains carrying our construct under different arabinose concentration. The measurement was carried out for 20 h. The x-axis shows the time of each measurement. The y-axis shows the fluorescence of mCherry in the arbitrary unit (AU). The blue data points represent the fluorescent intensity of p2547::mCherry strain, red data points represent the pBAD::mCherry strain. The arabinose concentration is shown at the top of each graph.](https://static.igem.org/mediawiki/2021/thumb/5/5c/T--Leiden--Measurement-Figure3.png/1600px-T--Leiden--Measurement-Figure3.png)

*Figure 5: The fluorescence intensity of strains carrying our construct under different arabinose concentration. The measurement was carried out for 20 h. The x-axis shows the time of each measurement. The y-axis shows the fluorescence of mCherry in the arbitrary unit (AU). The blue data points represent the fluorescent intensity of p2547::mCherry strain, red data points represent the pBAD::mCherry strain. The arabinose concentration is shown at the top of each graph.*

To improve the reliability of our results, we intended to use another constitutive promoter to calibrate pBAD. However, we didn't succeed to ligate it with mCherry by 3A assembly.

## Toxins

> **Aim: Determine which toxins are most effective in inhibiting cell growth.**
>
> ✔ Inducible promoter pBAD was successfully ligated to each toxin gene (see the [Parts Page](https://2021.igem.org/Team%3ALeiden/Parts)).
>
> ✔ Toxin CcdB and HOK exhibited inhibitory activity when adding 1% arabinose as inducers.
>
> ✔ The MazF and RelE showed no influence on *E. coli* TOP10 strain in liquid medium.
>
> ❔ Toxicity assay based on LB-agar plates may be another method to assess toxin lethality.
>
> ❔ Toxin may work better in minimal medium which resemble natural environment.

The toxicity of toxins is crucial for one function of the DOPL LOCK system: preventing HGT. We evaluated the inhibitory effects of four toxins on bacterial growth based on the constructs of pBAD::toxin. By adding arabinose inducers, we observed distinct inhibitory effects of the CcdB and HOK toxins. The toxins MazF and RelE didn't show influence on the growth rate of bacteria. We propose using minimal medium or LB-agar plates as alternative assessment approaches in the future.

![Figure 6The overnight growth curve of pBAD::CcdB (left) and pBAD::HOK (right) TOP10 strain under different arabinose concentrations. The high arabinose concentration was highlighted as red while the lower concentration was blue. It can be observed that the inhibitory effects increased as the concentration of arabinose increased.](https://static.igem.org/mediawiki/2021/0/06/T--Leiden--Results-Figure6.png)

*Figure 6: The overnight growth curve of pBAD::CcdB (left) and pBAD::HOK (right) TOP10 strain under different arabinose concentrations. The high arabinose concentration was highlighted as red while the lower concentration was blue. It can be observed that the inhibitory effects increased as the concentration of arabinose increased.*

## Antitoxins

> **Aim: Determine the expression level required for antitoxin to neutralize toxicity.**
>
> ✔ Constitutive promoter p162 ([BBa\_J23117](http://parts.igem.org/Part%3ABBa_J23117)) was successfully ligated to antitoxin CcdA and RelB(see the [Parts Page](https://2021.igem.org/Team%3ALeiden/Parts)).
>
> ❔ Toxicity assay with antitoxin cassettes.

The characterization of antitoxins could be done by co-transforming plasmids containing pBAD::toxin cassettes and constitutive promoter::antitoxin cassettes. The neutralizing effects of antitoxin could be assessed by gradually induced toxin gene expression. However, due to time restrictions, we only achieved ligation of p162 with CcdA and RelB by PCR-based cloning.

# Part assembly

## Creating the DOPL LOCK system

In the final stage of our lab time, we decided to refrain from balancing single TA systems due to limited lab time. Instead, We focused on creating the full DOPL LOCK system with constitutive promoters regulating both toxin and antitoxin genes. We successfully cloned the constructs of antitoxins p162::CcdA, p162::RelB, and of toxins p21::CcdB, p21::RelE by PCR-based cloning. However, 3A assembly of these constructs to the desired plasmid backbones didn't succeed.

# Discussion

In summary, we **characterized** the components of the DOPL LOCK system to achieve the **balance** of **TA gene expression** levels of TA systems. The characterization result provided us with information to select the **most compatible parts** for the full DOPL LOCK system. We successfully characterized all parts except the antitoxins in our experiments.

For the choice of Oris, our results showed that **p15A and pBR322/ROB** were the **most compatible** Ori pair, as they showed high levels of fluorescence when co-transformed. However, pSC101 and pUC have a low expression in RFP when co-transformed. The first version of the DOPL LOCK system was based on plasmids with the Oris p15A and pBR322/ROB.

For promoters, the **inducibility of pBAD** was first **analyzed** by ligating to the mCherry fluorescent protein cassette. Then it was **calibrated with the constitutive promoter p2547** successfully. The results could be applied to **predict appropriate inducer concentrations** to balance TA systems.

For the selection of the toxin, the **toxicity assay** showed that the **toxins CcdB and HOK exhibited significant inhibitory activity** on *E.coli* when induced by 1% arabinose. However, the toxins **MazF and RelE showed no effect** on the bacterial growth. Toxins tested in our experiment did not show instant kill effects, while previous research used them as kill switches [5]. There are several aspects that could explain these unexpected results. For instance, there is a possibility that the **antitoxin gene pre-existed** in our strains and neutralized the toxic effects as the four toxins we tested originated from *E. coli* [6]. Another possible explanation is that the strains already **gained resistance** to toxin before the expression of the toxin was induced. Future experiments could include **colony counting units** as an outcome measurement and/or use **minimal medium** to evaluate the toxic effect in alternative approaches. Furthermore, our [model](https://2021.igem.org/Team%3ALeiden/Model) shows the highest concentration of MazF reached is about 3 × 10-4 mM when 1% of arabinose is treated to bacteria, which is **not effective** based on the results from [CCU Taiwan in 2017](http://parts.igem.org/Part%3ABBa_K2292006). Using **constitutive promoters** instead of inducible promoters to regulate the MazF gene may ensure its toxicity on the cells.

Aside from modified methods, **including more TA systems** is also an important future aspect for the DOPL LOCK system. Toxins that act as nucleases, like HOK, are preferred as they prevent HGT further by degrading any DNA that could be taken in by environmental bacteria.

# References

1. https://blog.addgene.org/plasmid-101-origin-of-replication
2. All natural. (2007). Nature Chemical Biology, 3(7), 351-351. https://doi.org/10.1038/nchembio0707-351
3. Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., & Chu, J. (2016). A Guide to Fluorescent Protein FRET Pairs. Sensors (Basel, Switzerland), 16(9), 1488. https://doi.org/10.3390/s16091488
4. Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2016). 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nature Chemical Biology, 12(2), 82-86. https://doi.org/10.1038/nchembio.1979
5. Valenzuela-Ortega, M., & French, C. (2021). Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology. Synthetic Biology, 6(1). https://doi.org/10.1093/synbio/ysab003
6. Xie, Z., Qi, F., & Merritt, J. (2013). Development of a Tunable Wide-Range Gene Induction System Useful for the Study of Streptococcal Toxin-Antitoxin Systems. Applied and Environmental Microbiology, 79(20), 6375-6384. https://doi.org/doi:10.1128/AEM.02320-13
7. Yamaguchi, Y., & Inouye, M. (2011). Regulation of growth and death in Escherichia coli by toxin–antitoxin systems. Nature Reviews Microbiology, 9(11), 779-790. https://doi.org/10.1038/nrmicro2651

* [Introduction](#Introduction2)
* [Characterization of origin of replication](#Characterization_of_origin_of_replication)
* [Characterization of promoters](#Characterization_of_promoters)
* [Characterization of toxins](#Characterization_of_toxins)
* [Characterization of antitoxins](#Characterization_of_antitoxins)
* [Part assembly](#Part_assembly)

# Introduction

In our design, two TA systems are required to balance the expression of antitoxin and toxin. There are four critical components that determine the balance in this system, namely plasmid Ori, promoter in front of both toxin and antitoxin genes, toxin and antitoxin. To build up the system, we first collected multiple parts for each component and characterized them in a standardized protocol to determine which combination of all four components was the best option.

# Characterization of origin of replication

## RFP switch

To determine the compatibility of different Oris, we first obtained JUMP plasmids [1] from Addgene. This is a collection of plasmids designed by Valenzuela-Ortega et al., containing different Oris and sfGFP as the reporter [1].

We switched the already present sfGFP cassette of the JUMP plasmids to a mRFP1 cassette ( [BBa\_J04450](http://parts.igem.org/Part%3ABBa_J04450)). The products were transformed into *E. coli* DH5α and plated on agar plates containing kanamycin and spectinomycin. We isolated the plasmids from the successful transformants. Then, we restricted it with EcoRI & PstI and ran the gel electrophoresis (Supplementary Figure 1). The result showed that we successfully constructed four pJUMP plasmids with different Oris and the same RFP cassette.

Supplementary Figure 1

![Short description, in 3 words](https://static.igem.org/mediawiki/2021/f/f2/T--Leiden--Results-SF1.png)

*Supplementary Figure 1: The gel result of RFP switches. The first lane was the DNA ladder. The four lanes on the left were original pJUMP-4x plasmids with sfGFP as control (pJUMP46: 2311bp, 902bp; pJUMP47: 3039bp, 902bp; pJUMP48: 2508bp, 902bp; pJUMP49: 3039bp, 902bp). The other four lanes are pJUMP-4x plasmids with RFP cassette after switching (pJUMP46: 2311bp, 1110bp; pJUMP47: 3039bp, 1110bp; pJUMP48: 2508bp, 1110bp; pJUMP49: 3039bp, 1110bp). Some contamination was observed in pJUMP47-RFP.*

## sfGFP-RFP Cotransformation

Vectors containing sfGFP or RFP (listed in Table 1) were co-transformed into *E. coli* TOP10 in 16 combinations. Sixteen co-transformed strains and eight single-transformed strains (used as control) were cultured overnight for the measurement.

First, they were diluted to OD600 = 0.5 and transferred to 96-well plates for fluorescence measurement via plate reader (Spark, Tecan Group Ltd, Zürich, Switzerland).

*Table 1: The plasmids and their constructs used in the co-transformation*

| NAME | RESISTANCE | ORIGIN OF REPLICATION | INSERT | INCOMPATIBILITY GROUP | COPY NUMBER |
| --- | --- | --- | --- | --- | --- |
| pJUMP26 | Kanamycin | p15A | sfGFP | B | Medium/Low copy number |
| pJUMP27 | Kanamycin | pSC101 | sfGFP | C | Low copy number |
| pJUMP28 | Kanamycin | pUC | sfGFP | A | High copy number |
| pJUMP29 | Kanamycin | pBR322/ROB | sfGFP | A | Medium/High Copy number |
| pJUMP46 | Spectinomycin | p15A | RFP | B | Medium/Low copy number |
| pJUMP47 | Spectinomycin | pSC101 | RFP | C | Low copy number |
| pJUMP48 | Spectinomycin | pUC | RFP | A | High copy number |
| pJUMP49 | Spectinomycin | pBR322/ROB | RFP | A | Medium/High Copy number |

For the control groups with only one plasmid transformed, we surprisingly observed that the plasmid copy number only had minor impacts on the fluorescence (Figure 7). Minor differences caused by the plasmid copy number of Ori could be observed regarding RFP fluorescence (pUC > pBR322/ROB > pSC101 ≈ p15A). The sfGFP fluorescence of pSC101 and pUC showed much lower signals. Therefore, we focused on the RFP expression in the following data analysis.

![Figure 7 Fluorescence of single transformants with different Ori measured by plate reader. The left side shows sfGFP fluorescence from original JUMP plasmids.The right side shows RFP fluorescence of different pJUMP backbones inserted with RFP cassette.](https://static.igem.org/mediawiki/2021/9/98/T--Leiden--Results-Figure7.png)

*Figure 7: Fluorescence of single transformants with different Ori measured by plate reader. The left side shows sfGFP fluorescence from original JUMP plasmids.The right side shows RFP fluorescence of different pJUMP backbones inserted with RFP cassette.*

The co-transformation result showed p15A was highly compatible with pBR322/ROB. When compared with other co-transformation with pUC and pSC101 (Figure 8 and 9), they exhibited both high sfGFP and RFP fluorescence. We also discovered that plasmid combinations among incompatibility groups (shown in Table 1) may also generate poor signals. For instance, pSC101 showed poor RFP signals with all the other plasmids, despite it being the only plasmid from incompatibility C (Figure 10). Strains co-transformed with plasmids containing Ori pUC did not show any patterns related to incompatibility groups or plasmid copy numbers. (Figure 11).

![Figure 8 The fluorescence of single-transformed strains of pJUMP46-sfGFP and its co-transformants with other JUMP plasmids with RFP cassette.](https://static.igem.org/mediawiki/2021/8/8e/T--Leiden--Results-Figure8.png)

*Figure 8: The fluorescence of single-transformed strains of pJUMP46-sfGFP and its co-transformants with other JUMP plasmids with RFP cassette.*

![Figure 9 The fluorescence of single-transformed strains of pJUMP49-RFP and its co-transformants with other JUMP plasmids with sfGFP.](https://static.igem.org/mediawiki/2021/8/8c/T--Leiden--Results-Figure9.png)

*Figure 9: The fluorescence of single-transformed strains of pJUMP49-RFP and its co-transformants with other JUMP plasmids with sfGFP.*

![Figure 10 The fluorescence of single-transformed strains of pJUMP47-RFP and its co-transformants with other JUMP plasmids with sfGFP cassette.](https://static.igem.org/mediawiki/2021/2/2a/T--Leiden--Results-Figure10.png)

*Figure 10: The fluorescence of single-transformed strains of pJUMP47-RFP and its co-transformants with other JUMP plasmids with sfGFP cassette.*

![Figure 11 The fluorescence of single-transformed strains of pJUMP48-RFP and its co-transformants with other JUMP plasmids with sfGFP cassette.](https://static.igem.org/mediawiki/2021/4/4b/T--Leiden--Results-Figure11.png)

*Figure 11: The fluorescence of single-transformed strains of pJUMP48-RFP and its co-transformants with other JUMP plasmids with sfGFP cassette.*

In summary, the Ori pair of p15A and pBR322/ROB showed excellent compatibility (Figure 12). Both sfGFP and RFP fluorescence in co-transformed cells were even larger than the control. pJUMP26 and pJUMP49 were selected as the backbone of the DOPL LOCK. Whether these two plasmids both stably maintained in bacteria was verified by confocal microscopy (Figure 13). The microscopy imaging showed low heterogeneity of the culture. Most bacteria expressed both sfGFP and RFP.

![Figure 12 Fluorescence of co-transformed p15A and pBR322/ROB (in the middle) with their single-transformed control group (at the sides) with error bars. Green bars represent the sfGFP fluorescent signal while Red bars represent RFP signals.](https://static.igem.org/mediawiki/2021/1/11/T--Leiden--Results-Figure3.png)

*Figure 12: Fluorescence of co-transformed p15A and pBR322/ROB (in the middle) with their single-transformed control group (at the sides) with error bars. Green bars represent the sfGFP fluorescent signal while Red bars represent RFP signals.*

![Figure 13 The confocal microscopy image of the <em>E. coli</em> strain co-transformed with pJUMP49-RFP and pJUMP49-sfGFP. (A) The green fluorescence emission of the strain at 510 nm. (B) The red fluorescence emission of the strain at 608 nm. (C) The strain under normal light. (D) The merged RGB image of the green and red fluorescence.](https://static.igem.org/mediawiki/2021/d/d6/T--Leiden--Results-Figure13.png)

*Figure 13: The confocal microscopy image of the *E. coli* strain co-transformed with pJUMP49-RFP and pJUMP49-sfGFP. (A) The green fluorescence emission of the strain at 510 nm. (B) The red fluorescence emission of the strain at 608 nm. (C) The strain under normal light. (D) The merged RGB image of the green and red fluorescence.*

## sfGFP-mCherry Co-transformation

Even though we found the best combination of Oris in the sfGFP-RFP co-transformation assay, we also noticed the Förster resonance energy transfer (FRET) existed between two fluorescent proteins [2]. To avoid that, we performed experiments to substitute RFP with mCherry. However, the plate reader showed that the plasmid heterogeneity was present in strains transformed with plasmids containing pSC101 and pBR322/ROB (Figure 14). Due to time restriction, we stuck to previous results from the sfGFP-RFP co-transformation for the following experiment.

![Figure 14 Fluorescence of single transformants with JUMP plasmids with different Oris. The fluorescence was measured by the plate reader.](https://static.igem.org/mediawiki/2021/1/1e/T--Leiden--Results-Figure14.png)

*Figure 14: Fluorescence of single transformants with JUMP plasmids with different Oris. The fluorescence was measured by the plate reader.*

# Characterization of promoters

The major goal of our initial experiments is to balance the amount of toxin and antitoxin in bacterial cells. Ideally, all the toxin and antitoxin genes in our system should be regulated by constitutive promoters to prolong the state of balance. To do this, we calibrated inducible promoter pBAD ([BBa\_I0500](http://parts.igem.org/Part%3ABBa_I0500)) with constitutive promoter [BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100) (referred to as p2547 in the following). pJUMP49 plasmids containing pBAD::mCherry ([BBa\_K3962340](http://parts.igem.org/Part%3ABBa_K3962340)) and p2547::mCherry ([BBa\_K3962339](http://parts.igem.org/Part%3ABBa_K3962339)) were transformed into *E. coli* TOP10 strain. Their fluorescence under different L-arabinose concentrations was measured.

Supplementary Figure 2

![Short description, in 3 words](https://static.igem.org/mediawiki/2021/f/f9/T--Leiden--Results-SF3.png)

*Supplementary Figure 2: The gel result for plasmids involved in the inducibility test restricted with EcoRI and PstI. (A) The first lane shows the plasmid construct of pJUMP49-pBAD::mCherry (2964bp, 2096bp) and the third lane is the control plasmid pJUMP49-sfGFP (2964bp, 902bp). (B) The second lane shows the plasmid construct of pJUMP49-p2547::mCherry (2964bp, 922bp)*

## Calibration of pBAD with p2547

The result showed constant expression from p2547::mCherry and the expression from pBAD::mCherry increased gradually as the concentration of arabinose increased (Figure 15). The lowest inducibility was observed at the concentration of 0.00032% (w/v). We also concluded that pBAD is tightly regulated as it showed low leaky expression when no arabinose was added.

![Figure 15The fluorescence intensity of strains carrying our construct under different arabinose concentration. The measurement was carried out for 20h. Blue lines and dots were fluorescent of p2547 while red ones were from pBAD. The arabinose concentration was shown at the bottom of each graph.](https://static.igem.org/mediawiki/2021/thumb/5/5c/T--Leiden--Measurement-Figure3.png/1600px-T--Leiden--Measurement-Figure3.png)

*Figure 15: The fluorescence intensity of strains carrying our construct under different arabinose concentration. The measurement was carried out for 20h. Blue lines and dots were fluorescent of p2547 while red ones were from pBAD. The arabinose concentration was shown at the bottom of each graph.*

The relation between the arabinose concentration (w/v, log5) and the relative fluorescence of pBAD fitted nicely into a linear regression model with R2=0.9586 (Figure 16). From the regression, it could be calculated that at the arabinose concentration of 0.14% the transcriptional activity of pBAD is equal to p2547.

![Figure 16 Linear regression of arabinose concentration relative fluorescence of pBAD compared with p2547. The orange dash lines showed how p2547 related to pBAD transcriptional activity at the arabinose concentration of 0.14% (log<sub>5</sub>-1.22).](https://static.igem.org/mediawiki/2021/thumb/c/c1/T--Leiden--Measurement-Figure4.png/1600px-T--Leiden--Measurement-Figure4.png)

*Figure 16: Linear regression of arabinose concentration relative fluorescence of pBAD compared with p2547. The orange dash lines showed how p2547 related to pBAD transcriptional activity at the arabinose concentration of 0.14% (log5-1.22).*

*Detailed information about this part can be found on the [Measurement Page](https://2021.igem.org/Team%3ALeiden/Measurement)*.

# Characterization of toxins

In this project, we chose three type II TA systems (CcdA/CcdB, RelB/RelE, MazE/MazF) and one type I TA system (HOK/SOK) as candidates for DOPL LOCK. In type II TA systems, both toxin and antitoxin are proteins and the antitoxins act as antagonists to the toxin activities [3]. While in the type I TA system, the antitoxin is a small antisense RNA that base-pairs with the toxin encoding mRNA [4]. All antitoxins are degraded more rapidly than toxins.

## Ligation

To validate the toxicity, the toxin genes were first ligated to an inducible promoter pBAD and transformed into the TOP10 strain independently (all constructs are shown in the [Parts page](https://2021.igem.org/Team%3ALeiden/Parts)). The gel electrophoresis showed correct constructs of pBAD::toxin (Figure 17).

![Figure 17 The gel result for plasmids containing constructs of pBAD::toxin. These plasmids were restricted with EcoRI and PstI. (A) The second lane shows the plasmid construct of pJUMP27-pBAD::MazF (2947bp, 1718bp). The third lane shows the plasmid construct of pJUMP27-pBAD::HOK (2947bp, 1809bp). The fourth lane shows the plasmid construct of pJUMP27-pBAD::RelE (2947bp, 1676bp). (B) The second lane shows the plasmid construct of pJUMP27-pBAD::CcdB (2947bp, 1688bp).](https://static.igem.org/mediawiki/2021/c/ce/T--Leiden--Results-Figure17.png)

*Figure 17: The gel result for plasmids containing constructs of pBAD::toxin. These plasmids were restricted with EcoRI and PstI. (A) The second lane shows the plasmid construct of pJUMP27-pBAD::MazF (2947bp, 1718bp). The third lane shows the plasmid construct of pJUMP27-pBAD::HOK (2947bp, 1809bp). The fourth lane shows the plasmid construct of pJUMP27-pBAD::RelE (2947bp, 1676bp). (B) The second lane shows the plasmid construct of pJUMP27-pBAD::CcdB (2947bp, 1688bp).*

## OD assay

We first tested the overexpression of the toxins in DH5α strain. However, the DH5α strain metabolizes arabinose, which was used as the inducer of pBAD in our experiments. The induction of arabinose was not enough to induce bacterial death. Therefore, we switched to the *E. coli* strain TOP10 for the toxicity assay.

All strains containing the pBAD::toxin construct first grew overnight in LB medium, and were treated with different concentrations of arabinose at the exponential growth phase (OD = 0.5). Their ODs were measured by the plate reader overnight in intervals of 30 min. On the same plate, LB medium was used as blank control. Bacteria with no plasmids and bacteria containing the pBAD::mCherry construct were used as negative control. The bacteria treated with antibiotics were used as a positive control. Both strains were treated at the same arabinose concentration as toxin-producing strains.

From the plate reader results, the TOP10 control strain showed that the arabinose concentration used had no influence on cell growth. pBAD::mCherry strains showed cells treated with 1% arabinose had a lower OD value after 9 h (Supplementary Figure 3). This indicated that the high-level expression of proteins induced a burden to the bacterial growth. The result of toxin-producing strains showed that different toxins have different effects on bacterial growth. CcdB was the most promising toxin as it is the only one that inhibited bacterial growth significantly at the arabinose concentration of 0.5% (Figure 18). HOK also showed inhibitory effects but was less toxic than CcdB (Figure 19). Both toxins had higher inhibitory effects than pBAD::mCherry strain. Surprisingly, MazF and RelE showed no influence on bacterial growth (Figure 20 and 21).

Supplementary Figure 3

![Short description, in 3 words](https://static.igem.org/mediawiki/2021/9/9b/T--Leiden--Results-SF4.png)

*Supplementary Figure 3: The overnight growth curve of pBAD::RelE TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.*

![Figure 18 The overnight growth curve of pBAD::CcdB TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.](https://static.igem.org/mediawiki/2021/f/f1/T--Leiden--Results-Figure18.png)

*Figure 18: The overnight growth curve of pBAD::CcdB TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.*

![Figure 19 The overnight growth curve of pBAD::HOK TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.](https://static.igem.org/mediawiki/2021/7/76/T--Leiden--Results-Figure19.png)

*Figure 19: The overnight growth curve of pBAD::HOK TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.*

![Figure 20 The overnight growth curve of pBAD::MazF TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.](https://static.igem.org/mediawiki/2021/3/30/T--Leiden--Results-Figure20.png)

*Figure 20: The overnight growth curve of pBAD::MazF TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.*

![Figure 21 The overnight growth curve of pBAD::RelE TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.](https://static.igem.org/mediawiki/2021/c/c2/T--Leiden--Results-Figure21.png)

*Figure 21: The overnight growth curve of pBAD::RelE TOP10 strain under different arabinose concentrations. The high arabinose concentration is highlighted as red while the lower concentration is blue.*

One explanation for the low toxicity of MazF and RelE is that the bacteria proliferate rapidly in nutrient-rich medium and only essential metabolic pathways were triggered. Therefore, we tried to use M9 minimal medium for the toxicity assay. However, the optimization of the M9 minimal medium and the rest of this research line were abandoned due to time constraints. For further optimization of the experiments, this could be a focus for future experiments.

# Characterization of antitoxins

To investigate the antagonistic ability of the antitoxins, plasmids that constitutively expressed an antitoxin were required. Then, they could be co-transformed with the pBAD::toxin plasmids which had been cloned and validated above (Figure 22). By inducing different levels of toxin expression, the appropriate expression levels of antitoxin required to neutralize the effect of toxicity could be identified.

First, we used restriction enzyme cloning to ligate the constitutive promoters with the antitoxin. However, it turned out to be really difficult as the length of promoters we used were less than 100 bp. We also tried to increase the molar ratio between the promoter gene and plasmid backbone to more than 200:1, but didn't succeed either. To overcome this issue, we changed the cloning method to PCR-based cloning, in which the constitutive promoters were parts of the primers. We designed cloning primers (listed in the [Experiments page](https://2021.igem.org/Team%3ALeiden/Experiments)) and optimized the conditions of PCR including the primer concentrations and melting temperature. The gel electrophoresis showed successful ligation of p162::CcdA and p162::RelB (Figure 22) (see the [Parts page](https://2021.igem.org/Team%3ALeiden/Parts)). However, due to time limitations, we didn't perform toxicity assays with plasmids carrying antitoxin cassettes. The ligation products were directly used to create the full DOPL LOCK system.

![Figure 22 Gel electrophoresis results of p162::CcdA (second lane, 420 bp), p21::CcdB (third lane, 506 bp), p21::RelE (fourth lane, 494bp), and p162::RelB (fifth lane, 441bp).](https://static.igem.org/mediawiki/2021/b/b3/T--Leiden--Results-Figure22.png)

*Figure 22: Gel electrophoresis results of p162::CcdA (second lane, 420 bp), p21::CcdB (third lane, 506 bp), p21::RelE (fourth lane, 494bp), and p162::RelB (fifth lane, 441bp).*

# Part assembly

In our design, the inducible promoters were designed to be ligated with antitoxin genes as the function of a kill switch, and the constitutive promoters were designed to be ligated with the toxins. This construct can also be the tool to balance the single TA expressions. However, we only ligated the pBAD promoter with MazE and RelB by restriction enzyme cloning. Ligations of other constructs didn't succeed.

In the final stage of our lab time, we decided to skip balancing single TA systems. We tried to create the full DOPL LOCK system with constitutive promoters regulating both toxin and antitoxin genes. The constitutive promoters p162 ([BBa\_J23117](http://parts.igem.org/Part%3ABBa_J23117)) and p21 ([BBa\_J23113](http://parts.igem.org/Part%3ABBa_J23113)) were selected]. We successfully created the constructs of antitoxins p162::CcdA and p162::RelB (Figure 22), and toxins p21::CcdB and p21::RelE (Figure 22) via PCR-based cloning (see the [Parts page](https://2021.igem.org/Team%3ALeiden/Parts)). 3A assembly was performed to create constructs of pJUMP26-p162::CcdA-p21::RelE and pJUMP49-p162::RelB-p21::CcdB. No colony could be observed in the plates. The gel electrophoresis result showed the 3A assembly did not succeed.

# References

1. Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z., & Chu, J. (2016). A Guide to Fluorescent Protein FRET Pairs. Sensors (Basel, Switzerland), 16(9), 1488. https://doi.org/10.3390/s16091488
2. Brantl, S. (2012). Bacterial type I toxin-antitoxin systems. RNA Biology, 9(12), 1488-1490. https://doi.org/10.4161/rna.23045
3. Fraikin, N., Goormaghtigh, F., Melderen, L. V., & Margolin, W. (2020). Type II Toxin-Antitoxin Systems: Evolution and Revolutions. Journal of Bacteriology, 202(7), e00763-00719. https://doi.org/doi:10.1128/JB.00763-19
4. Valenzuela-Ortega, M., & French, C. (2021). Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology. Synthetic Biology, 6(1). https://doi.org/10.1093/synbio/ysab003
5. Zhang, S.-P., Wang, Q., Quan, S.-W., Yu, X.-Q., Wang, Y., Guo, D.-D., . . . He, Y.-X. (2020). Type II toxin–antitoxin system in bacteria: activation, function, and mode of action. Biophysics Reports, 6(2), 68-79. https://doi.org/10.1007/s41048-020-00109-8

# Discussion

For building up the DOPL LOCK system, the **major difficulty** lies in balancing the gene expression level of toxin-antitoxin (TA) systems. To achieve this, we first **characterized** the **influential parts** of TA gene expression, including the **origin of replication** (Ori) of the **plasmids**, **promoters**, **toxins** and **antitoxins**. The result of the characterization provided us with information to **select the most compatible parts** for the full system.

For the selection of the various **Oris**, our results show four Oris exhibited **high-level expressions** of GFP and RFP fluorescence **when transformed independently** (Figure 7). Regarding the plasmid with RFP, the fluorescent patterns were consistent with the previous characterization of JUMP plasmids, where **Ori p15A, pSC101** showed **lower signals** and **Ori pBR322/ROB** showed **moderate signals** [1]. However, the expression level difference between these Oris was much greater in the previous research. This could be because the bacterial strain DH5α, instead of the JM109 strain, was used in our experiment.

The compatibility of these Oris was evaluated by **co-transformation of two plasmids** with different reporter genes and the fluorescence of the co-transformants were measured. The results showed that **p15A** and **pBR322/ROB** were the **most compatible Ori pair**, while **pSC101** and **pUC** have **low expression** in RFP when co-transformed (Figure 8-11). The first version of the DOPL LOCK system was based on plasmids with the Oris p15A and pBR322/ROB. Their co-transformed strains showed high levels of both sfGFP and RFP fluorescence, even higher than the single-transformed strains.

Interestingly, incompatibility groups did not have a significant influence since we still **applied antibiotic selection pressure**. This phenomenon has also been observed by Velappan et al [2]. They showed that different plasmids containing identical Oris can be stably maintained in bacteria over 3 days without antibiotic selections [2].

To avoid the Förster resonance energy transfer (FRET) effect, we designed experiments to use the **sfGFP-mCherry fluorescent protein pair** instead of the sfGFP-RFP pair. However, we observed **heterogeneity** in plasmids with Oris pSC101 and pBR322/ROB from flow cytometry. A possible explanation is that **mutations** occurred in the fluorescent protein gene expression cassette. This mutation alleviates the expression burden, resulting in strains that are more competitive and will likely become the dominant phenotypes in the population.

It has been suggested that plasmid gene expression is not only related to **elements of the plasmid backbone** but also to the *E. coli* **host strain** and the **cultivation conditions** [3]. The plasmid copy number was not the only crucial factor of plasmid gene expression in our characterization experiment. Furthermore, the effect of different Oris on the plasmid copy number has not been well-investigated. For instance, the Ori pUC used in our experiment was generally regarded as a high-copy plasmid [4]. However, it was also reported to yield a lower copy number of up to 15 [3]. Therefore, **characterization** of Oris is **crucial** for plasmid gene regulation and should be focused on further experiments.

Additionally, the fluorescent proteins were very **stable** once formed [5]. In our experiments, they accumulated overnight and reached an equilibrium at around 9 h (Figure 15). This gene regulation of fluorescent proteins may not **resemble antitoxins** since most antitoxins were rapidly degraded. It would be interesting to use **fluorescent proteins with degradation tags** to simulate the antitoxin regulation. This would provide more information for selecting which parts to use. We would also like to test **more plasmid backbones** that are frequently used in **Standard European Vector Architecture** (SEVA) and **Biobricks** system to provide more flexibility to our system.

It has been suggested that the **combination of high expression of pBAD** and the **relatively low expression of the promoter** makes the pair **suitable** for studies of **TA systems**. We designed the same strategy to balance the TA systems. The pBAD (BBa\_I0500) was calibrated with p2547 (BBa\_J23100) for the selection of low expression promoters that regulate toxin genes. Our results show that when **0.14% L-arabinose** is added, **pBAD** will **express the same level** of protein as **p2547** (Figure 16). This result is in accordance with research of Santos et al., where the pBAD transcription activity is higher than p2547 at an arabinose concentration higher than 0.1% [6]. The calibration helped us **predict the appropriate promoters** that could be used to balance TA systems. Taking **CcdB/CcdA** as an example, the antitoxin–toxin **ratio greater than one** could prevent the **harmful effect** of CcdB in plasmid-containing bacteria [7]. We may predict that, in the TA system comprising **p2547::CcdB** and **pBAD::CcdA**, the **antitoxins** likely **neutralize toxic effects** when **a concentration higher than 0.14%** of arabinose is used.

For the selection of the toxins, the toxicity assay showed **CcdB and HOK exhibited significant inhibitory activity** on *E.coli* when induced by 1% arabinose (Figure 18-19). However, the **MazF and RelE showed no effect** on the bacterial growth (Figure 20-21). The toxins tested in our experiment **did not show** the **instant kill effects** as described in previous research, where they are used as kill switches. As the four toxins we tested originated from *E. coli*, there is a possibility that the antitoxin gene **already existed** in our strains and neutralized the toxic effects. It is also shown in literature that the TA system is **tightly regulated** in *E. coli*, as bacterial stress responses can trigger the transcription of chromosomally encoded TA systems without releasing active toxins [8]. The overexpression of toxins in our experiment may not exceed the regulation ability of the **pre-existing TA system**.

Another possible explanation for the low toxicity is that **resistant strains** had **already existed** in the culture before overexpression of the toxin was induced. The mutation for **escaping toxins is developed in 3 - 12 h** when the toxin is over-expressed, according to Garcia et al [9]. Here, they found that the mutations that inactivate the toxins were primarily in the −10 promoter region of the plasmids [10]. Other research showed that bacteria escaped biocontainment predominantly by inactivating mutations in the toxin genes [10]. As a single TA system could be inactivated easily by mutations, inclusion of **additional TA pairs** or multiple toxin genes should further **reduce the plasmid escape rate**.

Besides mutation, **the growth environment** is also an influential factor for toxicity. **CFU cell viability assays** are another **frequently used** method to evaluate how toxins influence **bacterial biofilm formation**. It has been shown by Kolodkin-Gal et al. that different TA systems affected bacterial cell death differently in the liquid medium and during biofilm formation [11]. In our human practices, [Nathan Fraikin](https://2021.igem.org/Team%3ALeiden/Human_Practices#Science) also suggested applying colony counting units (CFU) instead of optical density measured at 600 nm (OD600) to assess the toxic effects on biofilm formation in the future. It has been shown that induction of RelE or MazF transcription does not confer cell killing but instead induces a static condition in which the cells are still viable but unable to proliferate [12]. The bacteriostatic effects of these two toxins may only be observed via CFU cell viability assays. Therefore, the CFU cell viability assay is suggested as a **future experiment** in order to **clarify the effect** of the different toxins on cellular growth.

Additionally, when discussing our experiments with Prof. Dr. Dennis Claessen, he advised us to use **minimal medium** instead of LB-broth to test the toxin efficiency. This would **resemble the natural environment** much better, would the organism accidently escape from the biocontainment. It has been discovered that the ability of MazE to reverse the bactericidal effect of MazF was less effective in M9 minimal medium than in the rich LB medium [13]. However, additional nutrients need to be added to the minimal medium for different strains. Because of the limited lab time, we didn't succeed in **optimizing the components of the minimal medium**. Therefore, this could be an opportunity for further experiments to improve the toxin efficiency and characterization.

To gain more insights into the regulation of the toxin gene, we built a [model](https://2021.igem.org/Team%3ALeiden/Model) to investigate how arabinose influences the expression of pBAD::MazF. The model shows the **highest concentration of MazF** reached is about 3 × 10-4 mM when **1% of arabinose** is treated to bacteria. It is suggested that this concentration **is not sufficient for toxicity**, based on the results from [CCU Taiwan in 2017](http://parts.igem.org/Part%3ABBa_K2292006). After reaching the peak, the concentration of MazF decreases over time due to the **degradation of the arabinose**. Using **constitutive promoters** instead of inducible promoters to regulate the MazF gene may ensure its toxicity on the cells.

*All figures mentioned in the discussion panel can be found in the results panel.*

# References

1. Afif, H., Allali, N., Couturier, M., & Van Melderen, L. (2001). The ratio between CcdA and CcdB modulates the transcriptional repression of the ccd poison–antidote system. Molecular Microbiology, 41(1), 73-82. https://doi.org/https://doi.org/10.1046/j.1365-2958.2001.02492.x
2. Amitai, S., Yassin, Y., & Engelberg-Kulka, H. (2004). MazF-mediated cell death in Escherichia coli: a point of no return. Journal of Bacteriology, 186(24), 8295-8300. https://doi.org/10.1128/JB.186.24.8295-8300.2004
3. Chan, C. T. Y., Lee, J. W., Cameron, D. E., Bashor, C. J., & Collins, J. J. (2016). 'Deadman' and 'Passcode' microbial kill switches for bacterial containment. Nature Chemical Biology, 12(2), 82-86. https://doi.org/10.1038/nchembio.1979
4. Damalas, S. G., Batianis, C., Martin-Pascual, M., de Lorenzo, V., & Martins dos Santos, V. A. P. (2020). SEVA 3.1: enabling interoperability of DNA assembly among the SEVA, BioBricks and Type IIS restriction enzyme standards. Microbial Biotechnology, 13(6), 1793-1806. https://doi.org/https://doi.org/10.1111/1751-7915.13609
5. Fernandez-Garcia, L., Kim, J.-S., Tomas, M., & Wood, T. K. (2019). Toxins of toxin/antitoxin systems are inactivated primarily through promoter mutations. Journal of Applied Microbiology, 127(6), 1859-1868. https://doi.org/https://doi.org/10.1111/jam.14414
6. Jahn, M., Vorpahl, C., Hübschmann, T., Harms, H., & Müller, S. (2016). Copy number variability of expression plasmids determined by cell sorting and Droplet Digital PCR. Microbial Cell Factories, 15(1), 211. https://doi.org/10.1186/s12934-016-0610-8
7. Kolodkin-Gal, I., Verdiger, R., Shlosberg-Fedida, A., & Engelberg-Kulka, H. (2009). A Differential Effect of E. coli Toxin-Antitoxin Systems on Cell Death in Liquid Media and Biofilm Formation. PloS one, 4(8), e6785. https://doi.org/10.1371/journal.pone.0006785
8. Lee, C. L., Ow, D. S. W., & Oh, S. K. W. (2006). Quantitative real-time polymerase chain reaction for determination of plasmid copy number in bacteria. Journal of Microbiological Methods, 65(2), 258-267. https://doi.org/https://doi.org/10.1016/j.mimet.2005.07.019
9. LeRoux, M., Culviner, P. H., Liu, Y. J., Littlehale, M. L., & Laub, M. T. (2020). Stress Can Induce Transcription of Toxin-Antitoxin Systems without Activating Toxin. Molecular Cell, 79(2), 280-292.e288. https://doi.org/https://doi.org/10.1016/j.molcel.2020.05.028
10. Pedersen, K., Christensen, S. K., & Gerdes, K. (2002). Rapid induction and reversal of a bacteriostatic condition by controlled expression of toxins and antitoxins. Molecular Microbiology, 45(2), 501-510. https://doi.org/https://doi.org/10.1046/j.1365-2958.2002.03027.x
11. Tombolini, R., Unge, A., Davey, M. E., de Bruijn, F. J., & Jansson, J. K. (1997). Flow cytometric and microscopic analysis of GFP-tagged Pseudomonas fluorescens bacteria. FEMS Microbiology Ecology, 22(1), 17-28. https://doi.org/10.1111/j.1574-6941.1997.tb00352.x
12. Valenzuela-Ortega, M., & French, C. (2021). Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology. Synthetic Biology, 6(1). https://doi.org/10.1093/synbio/ysab003
13. Velappan, N., Sblattero, D., Chasteen, L., Pavlik, P., & Bradbury, A. R. M. (2007). Plasmid incompatibility: more compatible than previously thought? Protein Engineering, Design and Selection, 20(7), 309-313. https://doi.org/10.1093/protein/gzm005

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