Team:Leiden/Proof Of Concept



Proof of Concept

By combining the conclusions from our Engineering, we were able to develop a foundation for the proof-of-concept of DOPL LOCK. For our proof-of-concept, we have firstly determined which origin of replications (Oris) were most suitable for our system, which is important for the construction of our proposed double plasmid system. Secondly, we have established which of the toxins had the ability to kill the bacteria during our experiments. This also contributed to the proof-of-concept, since the final toxin antitoxin (TA) system constitutes the foundation of the biocontainment mechanism. These two components are critical building blocks for the design of our system. At the final stage of our proof-of-concept, we have been focusing on cloning a preliminary version of the DOPL LOCK. However, due to time constraints, we were unable to gather experimental data regarding the efficiency of our system. Therefore, we have developed future experiments that could be used to collect experimental evidence regarding (i) plasmid transfer and genetic stability of DOPL LOCK and (ii) the capability of our system to contain microbes to a specific location. Finally, we want to demonstrate how we envision DOPL LOCK as a standardized biocontainment platform and the ease of implementation in established plasmid nomenclature, such as the Standard European Vector Architecture (SEVA) [1].

Performed experiments

During our time in the lab we mainly focused on proving the two most important aspects of our biocontainment system in order to thereby show our proof-of-concept. This firstly includes the possibility of utilizing two independently replicating plasmids in one bacterium. Secondly, we evaluated the toxicity of the selected toxins to the bacteria. We consider the potency to kill and the stability of the system to be the most important criteria for a successful biocontainment system. By proving these pivotal building blocks of DOPL LOCK are effective, we can ensure a reliable foundation with high stability and the capacity to kill the bacteria for our final biocontainment system. This essential foundation proves the build-up of DOPL LOCK, as we designed it, is feasible with additional time.

  • Double plasmid - two independently replicating plasmids

    Since both plasmids need a different Ori, we needed to test the compatibility of different Oris to provide a stable and comparable expression of both plasmids in one cell. In our Engineering page we demonstrated that we were able to co-transform and stably express fluorescent proteins from both plasmids simultaneously in Escherichia coli (E. coli) TOP10 (Figure 1). By utilizing different Oris, we minimize the risk of plasmid segregation and unwanted recombination events due to plasmid incompatibility. As the data in our Results page show, we evaluated 16 different combinations of Oris in order to pick the right combination, ensuring a stable platform to test our toxin-antitoxin system. With this, we were able to show the stability of our designed double plasmid system, which is important to achieve our final proof-of-concept.

    Figure 1: The measured 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.

  • Toxicity of the TA systems

    One of the main criteria for a successful and effective biocontainment system is that the system is able to kill the bacteria under specified conditions. Once the right Ori combinations had been determined, we needed two effective TA systems that could be implemented for our proof-of-concept. We have validated four TA systems by assaying the cell death resulting from toxin induction, as described in our Engineering page. Here, we demonstrated that expression of the toxins CcdB and HOK is drastically impairing the growth of the bacteria as shown in our Results page (Figure 2). To conclude, these results indicate that these TA systems could be used to create a final DOPL LOCK that successfully induces lethality.

    Figure 2: 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.

Future experiments

With the two main criteria for the proof-of-concept now fulfilled, we designed the final stage of the proof-of-concept for DOPL LOCK. Unfortunately, we were not able to finalize the assembly of the entire system due to time constraints. However, with the acquired results, we propose experiments and further engineering to develop the full DOPL LOCK system and assess its effectiveness.

To develop our proof-of-concept further into the fully designed DOPL LOCK system, we have come up with a variety of experiments. First, to prove that the plasmid transfer of one of the plasmids leads to cell death and to assay the genetic stability of DOPL LOCK, we have proposed a design of these plasmids. As shown in Figure 3, the first plasmid contains the CcdB toxin and SOK antitoxin in the pJUMP29 backbone, with the pBR322/ROB Ori. The second plasmid contains the HOK toxin and the CcdA antitoxin in the pJUMP46 backbone, with the p15A Ori. Lastly, we've thought about experiments to further investigate the physical containment aspect of the DOPL LOCK system. We believe that these experiments will help turn our current proof-of-concept into a fully realised biocontainment system.

Figure 3: A schematic representation of the design for testing the proof-of-concept. The plasmid containing the pBR322/ROB Ori is expressing the CcdB toxin under the control of the promoter J23104 and the SOK antitoxin under the control of the promoter J23100. The plasmid containing the p15A Ori is expressing the HOK toxin under the control of the promoter J23104 and the CcdA antitoxin under the control of the promoter J23100.

  • Plasmid transfer

    To verify that both plasmids are present in the bacterium, we would need confirmation by isolating the plasmids from the bacteria. Subsequently, the plasmid DNA will be digested with BsaI and BlpI, which was determined by analyzing the plasmid sequences in silico. We found that BsaI can digest the DNA exclusively in the CcdB coding sequence, whereas BlpI can digest the DNA exclusively in the spectinomycin resistance gene. As a result, when both plasmids are present in the cell, two bands will appear on the electrophoresis gel (Figure 4).

    These same restriction enzymes could be used to eliminate either one of the plasmids from the DNA isolation, since we can linearize only one of the plasmids using these restriction enzymes. Via electrophoresis and gel extraction, the linearized and the intact plasmid can be separated. The intact plasmid can be extracted from the gel. This way, we can isolate and transform either of the plasmids separately and determine the potential of our proposed system to kill the bacteria when only one of the plasmids is present. As a final confirmation it will demonstrate that when both plasmids are in the cell, the bacteria survive and DOPL LOCK works as we intended.

    As a future goal we would like to simulate plasmid loss and substantiate that one plasmid can be lethal to the cell. We will do this by engineering an Eschericha coli bacterium with an inducible CRISPR/Cas9 integrated in the genome. This CRISPR/Cas9 system will be targeting both plasmids simultaneously and separately.

    Figure 4: The experimental workflow of validating the proof-of-concept and testing if the proposed system is working. Therefore, the plasmids isolated from the bacteria are digested with BsaI and BlpI for validation. To test the efficacy of the system, the plasmids will be digested with either BsaI or BlpI to separate both plasmids, extract them from the gel and transform them separately and together in E. coli.

  • Genetic stability

    To assay the genomic stability, we want to transform our DOPL LOCK system in the Escherichia coli strain Xl1-Red [2]. This E. coli strain has deletions in the mismatch DNA repair mechanism. Wild-type E. coli accumulates spontaneous mutations in the genome at a rate of 0.0025 per replication [3]. The E. coli strain Xl1-Red accumulates mutations 5000-fold faster than wild-type E. coli [2]. This strain could therefore provide us with a tool to assay the genetic stability of our proposed system over time. We can monitor the plasmid's integrity and stability with the assays described above and by sequencing the plasmids DNA [4].

  • Physical containment

    To simulate the escape of a GMO constrained by the DOPL LOCK system, we redesigned both plasmids in such a way that the antitoxins are conditionally expressed by the inducible promoter pBAD (BBa_I0500).

    Figure 5: A schematic representation of the design for testing the physical containment strategy. The plasmid containing the pBR322/ROB Ori is expressing the CcdB toxin under the control of the promoter J23104 and the SOK antitoxin under the control of the promoter pBAD. The plasmid containing the p15A Ori is expressing the HOK toxin under the control of the promoter J23104 and the CcdA antitoxin under the control of the promoter pBAD.

    To test how effective our DOPL LOCK system is in containing GMOs at a specific physical location, we would transform E. coli with the final plasmids represented in Figure 5. Subsequently, the bacteria are cultivated in LB medium with L-arabinose to ensure expression of both antitoxins, as elaborated in the Modeling and Measurement page. After homeostasis is achieved, the bacteria are plated on solid LB agar plates with varying amounts of L-arabinose, to simulate the gradient of the inducer around the physical area. Additionally, the bacteria are plated on LB agar plates without L-arabinose and on similar plates with glucose as shown in Figure 6. On these plates no colonies should be observed since the antitoxin should not be expressed anymore in the absence of the inducer or presence of glucose which suppresses the activity of the pBAD promoter. This will prove that our physical containment strategy is working and prevents DOPL LOCK contained organisms from wandering off in the wild.

    Figure 6: The experimental workflow for testing the physical containment strategy

The implementation

To be able to make the most impact with DOPL LOCK, we want it to be easy to implement and widely applicable. Therefore, our goal is to add DOPL LOCK as a part to the SEVA plasmid nomenclature. Our biocontainment modules will be added to the SEVA nomenclature as a potential substitute for the region of the plasmid containing the origin of transfer (OriT) and the antibiotic resistance gene. The OriT allows for conjugational mobilization of pSEVA plasmids into organisms for which no alternative transformation procedures are available [5]. Conjugation is one of the most well understood mechanisms of HGT [6]. By removing the OriT we could reduce the risk of HGT to protect the integrity of an ecosystem. With DOPL LOCK we want to eliminate this risk by eliminating the OriT and replacing this with our DOPL LOCK biocontainment module (Figure 7).

Additionally, we want to replace the antibiotic resistance cassette of the plasmids, with a split GFP as a selection marker of the presence of both plasmids (Figure 7). By using a split GFP selection marker the DNA coding for the subunit 11 of GFP is present on the plasmid harboring the gene of interest, while the other plasmid is harboring the DNA for the first 10 subunits of GFP. When both parts of the protein are expressed, GFP signals can be observed [8].

Figure 7: Proposed incorporation of DOPL LOCK in the SEVA nomenclature. Where the OriT is replaced with a biocontainment module and the antibiotic selection cassette can be replaced with a split GFP marker. This plasmid can be co-transformed with the plasmid harboring the split GFP and biocontainment module counter parts (not shown) to apply the biocontainment strategy.

With this proposed implementation, we want to provide a transparent and open-source platform that could be used for the application of SEVA plasmid-based GMOs in a semi-contained environment. These semi-controlled applications of GMOs entails applications in an environment where localized containment is possible, yet with a clear physical path for GMOs to spread into the environment, for example wastewater treatment plants, nitrogen fixation in plants or bioremediation of contaminated soil as described in our Implementation page.


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