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.
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.
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 . 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 . This would reduce the likelihood of horizontal gene transfer (HGT) drastically, unless the plasmid is transferred completely .
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 . 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 . There are over 27 incompatibility groups represented in the Enterobacteriaceae family and this is expanding since new genomic data is generated .
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  obtained via Addgene. These plasmids are designed according to the Standard European Vector Architecture (SEVA) nomenclature . 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.
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 .
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) according to 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.
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. The data of 3 biological replicates was analysed using R-studio and is presented in Figure 3 and are further elaborated in the Results.
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.
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 . Additionally, the SEVA 3.1 plasmids enable us to test more efficiently since the cloning is reversible . 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.
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 , the MazF/MazE TA-system , the HOK/SOK TA-system  and the CcdB/CcdA TA-system . 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), a ribosomal binding site (RBS) (BBa_J34801), the coding sequences of the toxins and a lambda T1 terminator (BBa_K864601), as shown in Figure 4.
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 Experiment 4. Subsequently, the genetic circuit is assembled using 3A-assembly according to Experiment 3: Biobrick ligation in the plasmid pJUMP26 . These vectors were transformed in the E. coli TOP10 strain according to 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 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.
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 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 . Therefore, we decided to develop an alternative protocol (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 . 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.
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.
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