Team:Wageningen UR/Results/Kill Switch

Xylencer

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Oscillating Kill Switch

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The goal of this subproject was to design a kill switch linked to a circadian oscillator. The whole genetic circuit would be incorporated into the Phage Delivery Bacterium (PDB). By linking the kill switch to a circadian oscillator, the PDB will have enough time to detect Xylella fastidiosa, if present in the environment, and initiate Xylencer phage proliferation. However, if X. fastidiosa is not present, the PDB will die due to our engineered kill switch. To be able to assess if the circuit works once introduced into the PDB (experiments developed in the model organism Escherichia coli), a green fluorescent protein was placed under the control of a circadian-responsive promoter. Simultaneously, the circadian-responsive promoter is being regulated by the cyanobacterial circadian oscillator core genes, which have been incorporated into the bacterium. GFP was shown to be expressed, increasing over time. Although, clear oscillations were not observed.

Introduction

Xylencer, which makes use of a Phage Delivery Bacterium (PDB) to carry the Xylencer phage genome as a plasmid, needs to have an induced lethality mechanism. The ideal mechanism for our system would be a kill switch which kills the PDB a few days after its release into the environment. The PDB should have some time to be able to detect Xylella fastidiosa if present. When present, the production of phages would kill the PDB. However, if X. fastidiosa is not present, the PDB should die through our engineered kill switch. For this, we hypothesize that a toxin-antitoxin system would work the best as kill switch for our system. Our hypothesis was based on a time-dependent accumulation of the toxin molecule which would surpass the levels of antitoxin deriving in an unbalance of the system and therefore in cell death. In order to achieve this time-dependent toxin accumulation, we decided to link the kill switch to a circadian oscillator.

Many organisms exhibit circadian oscillations in their physiological activities, with a period of ~24 h, as part of their adaptation to external environmental changes. These self-sustained oscillatory systems are found ubiquitously in both prokaryotes and eukaryotes [1]. In the cyanobacterium Synechococcus elongatus PCC 7942, the core circadian clock is composed of three components, KaiA, KaiB and KaiC, expressed as monocistronic kaiA and dicistronic kaiBC transcripts [2]. The kaiBC operon is upregulated by the KaiA protein and downregulated by the KaiC protein to generate a circadian oscillator [3]. KaiB can bind to KaiA, therefore opposing KaiA’s stimulatory activity by sequestering it, and to KaiC, driving the KaiC phosphorylation cycle in a clockwise direction (Figure 1) [4]. These changes drive corresponding oscillations in gene expression. The KaiABC system has previously been reconstituted in vitro [5]. These results suggested that the cyanobacterial oscillator could function when heterologously expressed in other organisms, which can have practical applications. Here, we try to reconstruct the Kai circadian oscillator in Escherichia coli, a well-studied model bacterium without a native circadian rhythm.

Figure 1. Mechanism of the cyanobacterial circadian oscillator core genes. KaiA upregulates the kaiBC operon, while KaiC downregulates it. KaiB can interact with KaiA sequestering it, and with KaiC promoting its autophosphatase activity.

As previously stated, an interesting practical application, herein explored, for this synthetic oscillator would be to link it to a kill switch. As a proof of principle, we used green fluorescent protein (GFP), to assess whether its concentration increases over time due to the oscillations.

  • Why do we want a kill switch? arrow_downward

    As synthetic biology makes advances producing real-world applications using genetically engineered microorganisms, the issue of biological containment becomes increasingly important. Approaches include inducing an auxotrophy for a particular metabolite, repressing the expression of an essential gene, or rewriting the genetic code to ensure dependency on a synthetic amino acid (see Biosafety Page). Kill switches are defined as artificial systems that result in cell death under certain conditions [8].

The system of choice will be the well-studied mazEF system, native from E. coli. There is an already existing part in iGEM (BBa_K2292006) which has previously been proposed and characterized by the iGEM17_CCU_Taiwan team. In our design, we propose to place the toxin, mazF, under the kaiBC circadian-responsive promoter, while the antitoxin, mazE would be under the control of a constitutive promoter (Figure 2).

Figure 2. Schematic of the design and construction of the mazEF kill switch system.

While the circadian oscillations are taking place, accumulation of transcribed protein will also occur, raising up levels over time. The concentration of toxin, which as previously stated will be under control of the kaiBC promoter, would increase while the concentration of antitoxin will remain constant over time. Consequently, at the very beginning of the circadian oscillations there will not be a sufficient accumulation of toxin to counteract the activity of the antitoxin, so the bacterium will be able to survive. After a few cycles elapsed, the concentration of toxin will be able to counteract the concentration of antitoxin, killing the bacterium (Figure 3).

Figure 3. Overview of the kill switch design concept controlled by the oscillations produced by the kai genes.
  • mazEF, our toxin-antitoxin system of choice arrow_downward

    The E. coli mazEF module was the first toxin-antitoxin system described as regulatable and responsible for programmed cell death. The product of mazF (MazF) is a stable toxin that inhibits translation by cleaving mRNA at specific sites. The product of mazE (MazE) counteracts the action of MazF, which on the contrary, is a labile protein. Prevention of MazF-mediated death requires the continuous production of MazE. [8].

Results

In order to make our kill switch work, and effectively kill the PDB, oscillations of GFP expression are expected approximately every 24h. Furthermore, increasing levels of fluorescence over time are also expected, meaning an increasing amount of transcribed GFP. Translated to our kill switch system, an increase in fluorescence will indicate increasing and accumulating amounts of toxin. Finally, a difference in fluorescence between the induced and non-induced cultures with rhamnose is also expected.

To further visualize the circadian oscillations, fluorescence assays were performed. Six different growing scenarios were tested.

  1. M9 minimal medium with 0.5% glycerol as carbon source.
  2. With the purpose to synchronize the population, cells were transferred to M9 minimal medium with no carbon source for 1 hour [9].
  3. M9 minimal medium, with the only difference that mineral oil was added to prevent evaporation during the fluorescence time courses.
  4. M9 minimal medium with 2% glycerol.
  5. M9 minimal medium with additional 0.5% succinate and 1mM leucine to promote slower growth and without induction to reduce population desynchronization [9].
  6. A scenario of a slowed down growth with mineral oil was combined.

In the scenario nº1, in which the bacteria were grown in M9 minimal medium (Figure 4), the replicates induced with rhamnose do show increasing levels of fluorescence over time. Despite this, similar increasing levels of fluorescence can be seen in the non-induced replicates.

Although the oscillations are not evident, this could be caused due to the visualization of GFP at population-wide level, instead of at single-cell level. Current studies show a better visualization of the oscillations when observed at single-cell level [10]. Unfortunately, and due to the lack of the required equipment, we could not perform those analysis.

Figure 4. Time course fluorescence output of the E. coli replicates grown in M9 minimal medium both induced with rhamnose (purple) and non-induced (orange).

In the scenario nº4, in which the bacteria were incubated for 1h with no carbon source, this way, synchronizing the population (Figure 5), a bigger difference in fluorescence between the induced and the non-induced replicates can be seen, being 2.5-fold higher. No real oscillations can be visualized, but this can again be explained due to observation techniques which, as previously mentioned, have been performed for a whole bacterial population, instead of only analyzing single dividing cells.

Figure 5. Time course fluorescence output of the E. coli replicates grown in M9 minimal medium, after synchronization of the population, of both induced with rhamnose (purple) and non-induced (orange).

Moreover, the graph above shows an increasing concentration of GFP over time, which is also of desire, since, as previously stated, we want an increase of toxin, until the point at which it can counteract the effect of the antitoxin and, kill the bacteria. Despite this, high fluorescence levels can also be seen in the non-induced one, revealing leakiness of the rhaBAD promoter. Leakiness of the rhaBAD promoter has also been seen by other team members. Diminishing leakiness of the system can be reached by randomization of the ribosome binding site (RBS). Promising results have been obtained which could also be implemented into this system, to circumvent the leakiness of the toxin-antitoxin system before it is implemented. All biological replicates showed this trend, although some variability in phase and amplitude. This is thought to be due to a lack of redundant mechanism, such as those present in cyanobacteria [11], to maintain robustness and synchronization. The proper control to assess the leakiness of the rhaBAD promoter, would have been a curve of fluorescence expression of GFP, driven only by the rhaBAD promoter without any regulation by the kai genes.

  • Other Scenarios arrow_downward

    In the scenarios nº 3 and 6, were mineral oil was used, GFP signal was not detected, since GFP requires oxygen for chromophore formation [12]. Together with scenarios nº2 and 5, these were finally not used for the analysis of the results and therefore data is not shown here.

Conclusion

We can conclude that the circadian promoter responds to the circadian oscillator genes introduced into the bacterium, therefore, producing increasing amounts of GFP. Even though GFP is being progressively produced, it is not clear if the production follows an oscillatory pattern. Better conclusive results might be obtained by analyzing single dividing cells.

  • Cloning Approach arrow_downward

    To reconstruct the oscillator, we constructed E. coli strains containing kaiABC in an operon under the rhamnose-inducible rhaBAD promoter (PrhaBAD) BBa_K3286216 (denoted as “kaiABC Operon Plasmid”). As well, a synthetic transcriptional reporter gene encoding green fluorescent protein (GFP) placed under the control of a circadian-responsive promoter from S. elongatus, kaiBC promoter (PkaiBC) (denoted as “Reporter Plasmid”) (Figure 6). Therefore, in the presence of rhamnose, the kaiABC operon will be expressed, activating the kaiBC promoter and, in turn, the GFP should exhibit oscillations.

    Figure 6. Plasmids built for the reconstruction of the circadian oscillator in E. coli. (a) Sequence map of the core oscillator components, kaiABC, expressed in an operon driven by a rhamnose-inducible promoter. (b) Sequence map of the GFP reporter driven by the circadian responsive promoter, PkaiBC.

    Several proteins including RpaA, RpaB and SasA have been shown to be implicated in the transmission of the Kai signal [6,7]. Ideally, an additional synthetic operon containing the previously mentioned genes under control of a different promoter (Figure 7), should also be constructed and incorporated into the aforementioned E. coli constructed strains.

    Figure 7: Sequence map of additional native cyanobacterial components, rpaA, rpaB, and sasA, expressed in a synthetic operon driven by a Tetracycline inducible promoter.
  • Construction of the Plasmids arrow_downward

    Primers for the amplification of the kaiABC operon from the genome of S. elongatus were designed, as well as primers for the amplification of the rhamnose and kaiBC promoter and GFP. Initially, construction of the different plasmids required for the synthetic circuit was approached using Golden Gate assembly. However, this method failed repeatedly in spite of our efforts to optimize the protocol conditions (e.g. more specific primers, addition of DMSO or GC enhancer). Consequently, we changed our strategy of cloning to Gibson Assembly, which fortunately, led to the right plasmids at the first attempt.

  • Experimental Approach arrow_downward

    In order to assess whether circadian oscillations take place when transplanted into a non-circadian organism, E. coli DH5⍺ strains were transformed with the two plasmids, kaiABC Operon Plasmid and the Reporter Plasmid, previously described. Unless otherwise stated, cells were grown at 37ºC overnight in 10 mL LB (Lysogeny broth) medium, supplemented with chloramphenicol and spectinomycin, at 25 and 50 µg/mL, respectively. Single colonies were used to inoculate cultures into M9 minimal medium with 10 g/L tryptone, 0.5% glycerol, antibiotics and 3.75 mM rhamnose induction, as necessary.

    The fluorescence assays were performed in a microplate reader (SynergyTM Mx BioTek) using 96-well plate. Cells grown as described above were washed twice in M9 minimal medium during the LB-to-M9 transition in order to eliminate LB traces, which could have an impact on the GFP measurement. Cells were resuspended to an OD600 (optical density at 600 nm) of ~0.2 and grown at 37ºC in 96-well plates in 200 µL of M9 minimal medium with different conditions. The optical density (OD600 nm) readings of the 96-well plates were normalized to values for 1 cm pathlength (Spectrophotometer DiluphotometerTM IMPLEN). The course time for monitoring the different growth curves was set at 72 h. Biological and technical triplicates were included.

  • References arrow_downward
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    9. Amitai, S., Yassin, Y., and Engelberg-Kulka, H., (2004) MazF-Mediated Cell Death in Escherichia coli: a Point of No Return. Journal of Bacteriology 186(24), 8295-8300.
    10. Chen, AH., Lubkowicz, D., Yeong, V., Chang, RL., and Silver, PA. (2015) Transplantability of a circadian clock to a noncircadian organism. Sci. Adv. 1, e1500358.
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