Part:BBa_K3900053
pBad:Trz:dTer:ssTNT.R3:araBAD:PrompC:GFP:T1
signalling cascade for TNT detection (bacteria)
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
Illegal BamHI site found at 3725 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2102
Illegal NgoMIV site found at 2224
Illegal NgoMIV site found at 2354
Illegal AgeI site found at 495
Illegal AgeI site found at 3560 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4542
Illegal SapI site found at 3542
Source organism
Escherichia coli, Aequorea victoria
Biological origin
Two-component systems are found in eukaryotes and prokaryotes and allow sensing of environmental conditions in a stimulus-response mechanism. Since two-component mechanism can be found in plants and bacteria and is well conserved throughout various species, it is feasible to engineer chimeric two component systems. Our signaling cascade in plants is based on a two-component system which is involved in chemotaxis of Escherichia coli (E. coli). Chemotaxis is a complex mechanism that allows cells to sense and move towards attractants or move away from repellents. The first step of the complex signaling during the chemotaxis is the binding of the ligand, for example a carbon source such as ribose or glucose, to a specific receptor located in the periplasm, the periplasmic binding protein (PBP). PBPs are homologous proteins, that consist of two globular domains and a connecting hinge region. Upon binding of a ligand, a drastic conformational change is induced. The so-called “Venus-flytrap”-mechanism enables embedding of the ligand between the globular domains and allows the binding of the periplasmic binding protein by membrane-bound receptors that are specific for each PBP [1]. In the case of RBP, as well as the galactose/glucose binding protein (GBP), the membrane bound receptor involved in the signaling cascade is the Trg receptor. This receptor type officiates as a homodimer and consists of a periplasmic ligand-binding domain, a helical transmembrane segment and a helical cytoplasmic region [2]. The cytoplasmic part of the receptor serves as a framework for a super-molecular receptor-kinase signaling complex. In this complex the histidine kinase CheA is bound to the receptor. The binding is facilitated by a conformational change upon PBP binding to Trg [3]. The activity of the kinase on the other hand is downregulated upon ligand binding by Trg, which in turn inhibits the activity of the two response regulators CheB and CheY [3]. CheB is a methylase that acts antagonistic to the methylation enzyme CheR. CheR methylates Trg and makes it less responsive to the binding of the ligand, while CheB reverts this reaction, therefore increasing sensitivity to a ligand. Phosphorylated CheY docks to the flagellar motor protein and induces a tumbling state [3]. The inhibition of the activity of both CheB and CheY by the binding of the ligand results in a stable forward swimming state along an increasing attractant gradient.
The signaling cascade
Our signaling cascade is depicted in figure 1. The two-component system of the chemotaxis was engineered to allow recognition of a ligand of interest as a stimulus and result in a visible output (GFP). The engineered signaling cascade is based on a computationally evolved RBP, that binds to Trinitrotoluene (TNT). Baumgartner et al. first integrated the downstream chemoreceptor Trg to the osmosensor EnvZ in 1994 [4]. The resulting Trz fusion protein was composed of the periplasmic and the transmembrane domain of the chemotaxis receptor Trg which was linked via the HAMP-domain with the cytoplasmic part of the histidine kinase EnvZ [4]. Both parts of the Trz1 protein are derived from E. coli and combine the ability of recognizing ligand-bound RBPs of Trg with capability to phosphorylate the transcription factor OmpR upon contact with the RBPs. The resulting signaling cascade can detect the RBP-bound ligands. As a consequence of detection, expression of genes that are controlled by the OmpC promoter, which is activated by phosphorylated OmpR is induced [5, 9]. In the past it has been attempted to alter the binding pockets of PBPs to allow detecting of other ligands, and thus turn E.coli into a biosensor for various small molecules, while maintaining the same signaling cascade and reporter output downstream [6, 7]. This approach has been successfully transferred from bacteria onto plants by the group of Medford in 2011 [8]. In our project, we aimed at re-designing this signaling cascade in plants, to make it suitable for the detection of chemical weapons and their precursors in a natural environment. The signaling cascade poses as a powerful and diverse tool that could be designed by de novo and constructed by the means of synthetic biology and be applied in a wide range of applications ranging from biotechnology to biomedicine. Projects from future iGEM-teams can benefit from this part.
Tests with endogenous receptors
A pSB1C3 plasmid with the signaling cascade was cloned into E. coli DH5α. Since the TNT receptor is not translocated to the periplasmic space, it was tested whether the endogenous RBP/GBP expression is sufficient for activating the signaling cascade. A preliminary growth experiment was conducted with eight positive clones. The clones were inoculated with an optical density (OD) of 0.1 in 15 mL LB media in Erlenmeyer flasks and grown at 37°C at 180 rpm in an incubator. After 2 hours 0.2% (0.013 M) arabinose was added. After additional 4 hours, ribose (20 mM) was added. Liquid cultures were subsequently grown overnight. The liquid cultures were then centrifuged. For six out of 8 cultures, a greenish color in the pellets was observed.
A second growth experiment was carried out under the same conditions as described above, with the exception that other ligands were supplemented. Since E. coli favors glucose over ribose as a carbon source, we concluded that the GBP may have a higher affinity to Trz and, therefore, may be the more effective inductor of the signaling cascade. Glucose was added at a concentration of 20 mM and 200 mM to evaluate whether a higher concentration leads to more GFP expression. Additionally, we tested the influence of the changes in osmolarity by adding different amounts (50%/25%/12.5% w/v) of polyethylene glycol 4000 (PEG4000) without prior induction. PEG4000 is a chemically inert polymer, that is not metabolized by E. coli and is therefore well suited as osmolyte. A control run was carried out with only arabinose to rule out, that arabinose is sufficient to activate the signaling cascade. Further experiments were performed with the ligand and without arabinose induction, to rule out the osmotic pressure from the ligand addition as a contributing factor.
The liquid cultures were pelleted by centrifugation. Several pellets appeared greenish while the negative control lacked a greenish color. The gradation was the following from greenest to palest:
1. Arabinose + Ribose
2. Arabinose + Glucose (20 mM) / Arabinose + Glucose (200 mM)
3. PEG4000 (50% w/v) / PEG4000 (25% w/v)
The other pellets, except for the negative control also displayed a greenish color but were not differentiable by eye. To verify, that the green color was due to increased GFP expression, we used confocal laser scanning microscopy (CLSM) (Figure 2). The pellets were dissolved in 1 mL PBS pH 7.2, and random spots were examined. The resulting histograms were plotted with their fluorescence intensities against their frequencies (Figure 3).
It appears that induction by arabinose + ribose leads to the highest GFP expression. The induction by osmotic stress does not seem to differ drastically from the base fluorescence of the negative control, therefore indicating, that in fact the signaling cascade is expressed and contributes to the fluorescence. The results also suggested that ribose is more suitable for following experiments since it seems to lead to higher induction of the signaling cascade. Furthermore, the overall low fluorescence activity may stem from the low expression of RBP, the starting point of the signaling cascade. The next step was the overexpression of RBP to increase the initial stimulus, that consequently leads to a higher output signal.
Overexpression of endogenous receptors
We decided to co-transform the pSB1C3 plasmid containing the signaling cascade a plasmid containing a RBP, that was constructed for our library experiments. Furthermore, the fact that TNT-receptor remained in the signaling cascade did not pose as a problem, as it is not exported into the periplasmic space and does not influence the intracellular signaling. The plasmids were co-transformed into E. coli BL21(DE3). After heat shock transformation and subsequent over-night growth on the LB-agar plates, biological triplicates were picked, grown in 15 mL LB-media at 37°C over-night in Erlenmeyer flasks. The remaining 5 mL of each culture were used for a preliminary growth experiment to assess which culture has the highest fluorescence after induction with arabinose. The colonies were again inoculated at an OD of 0.1 in LB-medium. After 2h, arabinose was added, after 4h IPTG (1 mM) was added and after 6h ribose was added. The cultures were grown over-night and centrifuged, where the pellet of the three replicates appeared in the same greenish color.
The next growth experiment was conducted to quantify whether the additional overexpression of RBP leads to higher fluorescence. Addition of arabinose, IPTG and ribose to the media was performed as for the preliminary experiment. One strain without RBP overexpression and one strain with RBP overexpression were tested with the same combinations of additives. No biological replicates were used in this experiment. The liquid cultures were diluted to OD 2 and 300 µL of each culture were transferred thrice to a 96 well plate. GFP fluorescence was measured in the Tecan infinite M200 microplate reader (from now on called Tecan reader). Each well was measured in 9 technical replicates at overlapping but different spots. The mean of the technical replicates was calculated automatically by the manufacturer’s software. The means and the standard deviations are shown in figure 4. The fluorescence of the background of the LB-medium was subtracted. In general, we demonstrated in our experiment, that the GFP expression increases with RBP overexpression, showing that the endogenous RBP expression is a bottleneck in the signaling cascade. Induction with arabinose and subsequent addition of ribose leads to the highest fluorescence. The ratio of the cultures induced and supplemented with ribose relative to the negative control increased greatly in RBP overexpressing strains. This indicates that the overexpression does in fact increase the stimulus of the signaling cascade and further validates, that the signaling cascade is working. IPTG does not show a positive effect on the signaling cascade. Possible explanations to this observation include that the lac-promotor is leaky to uninduced expression or that IPTG induction leads to formation of inclusion bodies. The pellets from other cultures, with exception of the negative control, also demonstrated a greenish color, however not differentiable by eye.
Overexpression of computationally designed receptors
After the successful implementation of the experiments towards establishing the signaling cascade with RBP overexpression, we decided to overexpress our computationally designed diisopropyl methylphosphonate (DIMP) and 1,3,5-benzenetricarboxylic acid (BTCA) receptors for the purpose of testing them in-vivo. We used vectors pRSETB-DIMP-receptor/BTCA-receptor for expression of the receptors for in-vitro testing, were used for a co-transformation with the signaling cascade in BL21(DE3). After heat shock transformation, colonies were picked and cultivated in 15 mL LB-medium and grown under the same conditions as before. A growth experiment was conducted. Cells were grown in 1 mL LB in a 96-deep well plates at 37°C on a rotary shaker at 500 rpm. Additives were added in the same manner as before. Three biological replicates were tested. Fluorescence was measured in 96-well plates in the Tecan-reader. For each well, nine measurements were conducted, and cells were diluted to an OD of 1. The fluorescence of the LB-medium was subtracted. All three replicates show the same trends in fluorescence levels. The sole addition of either arabinose or BTCA in various concentrations led to fluorescence levels similar or below the fluorescence levels of the negative control (Figure 5). This finding indicates no significant activation of the signaling cascade. Addition of arabinose and BTCA led to significantly higher fluorescence levels, with the culture exposed to the highest concentration demonstrated the highest fluorescence. Addition of 1% ethanol to the cultures grown on arabinose, also increased fluorescence in comparison to the negative control. This can be explained by the osmotic pressure from ethanol, which activates the osmotic stress responsive promotor. However, the addition of the ligand only did not result in higher fluorescence and the arabinose supplementation is not sufficient for inducing osmotic stress, it is likely, that the signaling cascade is the main contributor to the higher fluorescence levels. This provides evidence for the receptor binding the ligand. This conclusion is in line with the results of the in-vitro testing and is a proof of concept for the computational receptor design of the receptor binding specifically to the BTCA. To provide further evidence, yet another growth experiment was done to compare the abilities of the BTCA-receptor and RBP to activate the signaling cascade. The DIMP-receptor showed no affinity to DIMP nor ribose (data not shown). Possible ways to obtain a functional DIMP-receptor are computational re-design, or mutation of RBP by Darwin assembly. Our attempt toward construction thereof can be found in description of our library experiments.
3D deconvolution Widefield Fluorescent Microscopy and super resolution 3D structured illumination microscopy
Lastly, we performed microscopy experiments to detect the GFP induced by the signaling cascade on a single cell level. For this purpose, we had access to a Deltavision OMX V4, a cutting-edge life cell imaging microscope, which uses Fourier transformation to gain a resolution higher than the diffraction limit of visible light. We did 3D deconvolution Widefield Fluorescent Microscopy and super resolution 3D-structured illumination microscopy. The BTCA-receptor culture supplied with BTCA and the respective negative control were used in this experiment. Figure 6 shows the 3D deconvolution widefield fluorescent microscopy, the differential interference contrast channel and the eGFP channel and the merged overlay of both channels in various magnifications. Evidently, the BTCA supplemented BTCA-receptor culture displayed a GFP fluorescence while the negative control showed none.
The results of the super resolution 3D-structured illumination microscopy can be seen in figure 7. The differential interference contrast channel, the eGFP channel, and the merged overlay of both channels in different magnifications are shown. The super resolution 3D-structured illumination microscopy confirms once again that we obtain a green fluorescence after arabinose induction and addition of BTCA to the culture, while the negative control showed no fluorescence.
References
- Borrok, M. J., Zhu, Y., Forest, K. T. & Kiessling, L. L. Structure-based design of a periplasmic binding protein antagonist that prevents domain closure. ACS chemical biology 4, 447–456 (2009).
- Antommattei, F. M., Munzner, J. B. & Weis, R. M. Ligand-specific activation of Escherichia coli chemoreceptor transmethylation. Journal of bacteriology 186, 7556-7563 (2004)
- Falke, J. J., Bass, R. B., Butler, S. L., Chervitz, S. A. & Danielson, M. A. The two-component signaling pathway of bacterial chemotaxis: a molecular view of signal transduction by receptors, kinases, and adaptation enzymes. Annual review of cell and developmental biology 13, 457–512 (1997).
- Baumgartner, J. W. et al. Transmembrane signalling by a hybrid protein: communication from the domain of chemoreceptor Trg that recognizes sugar-binding proteins to the kinase/phosphatase domain of osmosensor EnvZ. Journal of bacteriology 176, 1157–1163 (1994).
- Tavares, D. Changing the ligand-binding specificity of E. coli periplasmic binding protein RbsB by rational design and screening. University of Lausanne 2020.
- Tavares, D. et al. Computational redesign of the Escherichia coli ribose-binding protein ligand binding pocket for 1,3-cyclohexanediol and cyclohexanol. Scientific reports 9, 16940 (2019).
- Antunes, M. S. et al. Programmable ligand detection system in plants through a synthetic signal transduction pathway. PloS one 6, e16292 (2011).
- Looger, L. L., Dwyer, M. A., Smith, J. J. & Hellinga, H. W. Computational design of receptor and sensor proteins with novel functions. Nature 423, 185–190 (2003).
- https://www.uniprot.org/uniprot/P0AA16.
| None |