Phosphate Uptake
In native Bacillus subtilis, phosphate inducible promoter PpstS BBa_K4418004, is repressed in increasing phosphate concentrations. For application in phosphate remediation where phosphate concentrations in solution are elevated, such as in eutrophic rivers or wastewater, this would mean the expression of any phosphate importers used to uptake phosphate would be inhibited as phosphate levels are in excess. As a result, the regulation of the PpstS promoter obeys the logic of a NOT gate, where the presence of an input (high phosphate) causes an output of 0 (luciferase), and the absence of an input (low phosphate), causes the output to be 1.
To allow expression of phosphate importers to in the presence of high phosphate for environmental remediation, we DESIGNED a NOT gate to ensure the expression of phosphate uptake systems in the presence of high phosphate. A schematic of this is shown in Fig. 1A-B. As shown in Fig.1A, in the presence of high phosphate, the PpstS promoter is repressed, such that the expression of a downstream repressor can no longer take place. As a result, the cognate promoter of the repressor (Prepressor) becomes active and can allow for the expression of phosphate importers in the presence of high amounts of phosphate. When the levels of phosphate deplete, the PpstS promoter becomes active, allowing for the expression of the repressor which subsequent represses transcription of a downstream phosphate uptake system. This proposed design ensures phosphate uptake only occurs when phosphate levels in solution are elevated.
Figure 1. Design of a phosphate regulated NOT gate. A) Regulation of the phosphate regulated NOT gate in phosphate replete. High phosphate conditions allow for the repression of a repressor driven by the promoter PpstS. With the repressor no longer made, the cognate promoter of this repressor can allow for transcription of phosphate uptake genes to import external phosphate. Note, the NOT gate section of the circuit is boxed in light blue. B) Regulation of the phosphate circuit in the phosphate deplete conditions. The presence of phosphate causes activation of transcription from PpstS and subsequent expression of a repressor which is able to repress its’ cognate target promoter and expression of phosphate uptake genes.
Functionality of the heterologous NOT gate in Bacillus subtilis
For the characterisation of the NOT gate, we initially wished to use the PxylA promoter (BBa_K1351039) to drive the expression of the heterologous repressor SarA (BBa_K4418005) of Staphylococcus aureus, the target promoter of which is the PsprC (BBa_K4418006)[1]. The use of the PxylA promoter was favoured to first assess as to whether the circuit functions prior to testing the final design using our characterised PpstS promoter. A full schematic of the circuit is shown in Fig. 2. In this circuit design, the expression of the regulator SarA is induced by the promoter PxylA in the presence of xylose, and subsequently translated by the consensus B. subtilis RBS (BBa_K090505). To prevent read-through from PxylA, a strong transcriptional terminator BBa_K4418007 is used. The resulting circuit should show a reduction in luxABCDE in the presence of xylose through the composite part BBa_K4418008.
Figure. 2 Schematic of the PxylA inducible NOT gate ( BBa_K4418008). In the proposed xylose inducible SarA based NOT, the presence of xylose allows for the expression of the repressor SarA from PxylA which leads to subsequent repression of the PsprC promoter and luxABCDE activity. Boxed in blue is composite part comprising the xylose-inducible NOT gate, part BBa_K4418008.
We BUILT the circuit shown in figure 2 using Golden Gate cloning, assembling the NOT gate parts together and then assembling it into the plasmid pBSANDlux which contained our luciferase reporter (luxABCDE). Subsequently we transformed the assembled plasmid into Escherichia coli DH5α to test for successful integration by visible white colonies, indicating replacement of the RFP cassette in pBSANDlux by our plasmid. We then isolated transformed colonies, extracted our plasmid and transformed it into Bacillus subtilis, our chassis, for validation.
We TESTED our circuit by exposing our transformed Bacillus subtilis to increased xylose concentrations, measuring the relative luminescence units (RLU) with a plate reader, normalised by the optical density (OD 600) of the cultures. This measurement inform us of the relative promoter activity for a given concentration of cells.
Following xylose induction, we LEARNT that SarA is able to repress its cognate promoter PsprC which drives the expression of a luciferase reporter (luxABCDE). As a result, luciferase output shown decrease as a function of increasing xylose concentrations. As shown in Fig. 3, consistent with our theorised design, the repression of luciferase activity occurs in a xylose dependent manner with basal activity of 293025 RLU/OD600 units with output reduced to 40703 RLU/OD600 (7.2 -fold repression) in the presence of 0.2 % xylose.
Figure 3. Activity of the xylose inducible NOT gate. Promoter activity of PsprC in response to xylose inducible regulation of the repressor SarA, expressed as luminescence in RLU/OD600. Data show mean values and ± SD of a single biological replicate carried out using duplicate technical measurements.
Phosphate Release
Bacillus subtilis contains the gene encoding for thephosphodiesterase glpQ and the phosphatase encoding gene phoD ( BBa_K4418001) both of which are activated under phosphate starvation are expressed and active in the degradation of teichoic acid in the cell wall. This leads to the release of phosphate as this is where it is natively stored in Bacillus subtilis. The phosphate released from this mechanism is then utilised by the bacteria for its metabolic processes.
For being used in a phosphate release circuit that induced by a plant signal released under phosphate stress, we DESIGNED a circuit in which an inducible promoter PmaeN BBa_K4418000 was inserted upstream of both genes. As shown in Fig. 4 PmaeN is induced by malate, an exudate released by plant roots when lacking in phosphate. As a result, phoD and glpQ will mediate the release of phosphate induced by malate i.e. when plants find themselves in need of phosphate. In this manner, plants will have a controlled supply of phosphate, one that is activated upon demand and that will not lead to excess phosphate levels in soil.
Figure. 4 Circuit schematic of the phosphate release circuit BBa_K4418003. Induction with malate allows transcriptional activation from the PmaeN promoter, allowing for expression of the genes glpQ and phoD which encode Wall Teichoic Acid (WTA) degrading enzymes. Expression of both enzymes allows for phosphate release from WTA degradation.
Testing the inducible promoter
The characterization the PmaeN promoter was carried out through the insertion of the promoter into the luciferase reporter plasmid pBSGGlux. A schematic of the circuit ( BBa_K4418003) is show in Fig. 5 in which malate induction activates the MalK protein kinase that phosphorylates the PmaeN response regulator MalR. This would lead to luxABCDE expression which would then be measured through the emitted bioluminescence. Therefore, the circuit would emit a measurable signal upon malate induction allowing us to verify PmaeN functionality.
Figure. 5 Schematic of the PmaeN – luxABCDE biosensor in B. subtilis. The malate responsive promoter PmaeN is regulated by the MalKR Two-Component System. In this circuit design, malate induction causes activation of the sensor kinase MalK, leading to subsequent phosphorylation of the response regulator MalR and activation of the PmaeN promoter, resulting in measurable luxABCDE expression.
We BUILT the circuit shown in Fig. 5 using Golden Gate cloning, assembling PmaeN into the plasmid pBSGGlux containing luxABCDE. Subsequently we transformed the assembled plasmid into Escherichia coli DH5α to test for successful integration by visible white colonies, indicating replacement of the RFP cassette in pBSANDlux by our plasmid. We then isolated transformed colonies, extracted our plasmid and transformed it into B. subtilis, our chassis, for validation.
We TESTED our circuit by exposing our transformed B. subtilis to increased malate concentrations, measuring the relative luminescence units (RLU) with a plate reader, normalised by the optical density (OD 600) of the cultures. This measurement informed us of the relative promoter activity for a given concentration of cells.
We then LEARNT that the construct the luminescence emission occurred in a dose dependant manner upon malate induction (Fig. 6). This allowed us to verify the suitable working range of this promoter and, furthermore, to utilize it in our designed release circuit
Figure. 6 Dose response curve of the PmaeN-luxABCDE reporter. Cells harbouring the integrated PmaeN-luxABCDE circuit were grown to mid-exponential phase (OD600 = 0.1) in MCSE medium and challenged with various concentrations of malate shown. Promoter activity following malate induction are expressed as luminescence in RLU/OD600 of 35-45 minutes post induction. Note, due to the log-scale on the x-axis, values at 0.001 mM represent basal (uninduced) activity. Values presented show mean and ± standard deviation of triplicate measurements performed on three different days (n = 9).
Testing the Release Circuit
Upon characterizing the inducible promoter PmaeN. We then proceeded to BUILD the release circuit containing glpQ and phoD (Fig. 4). Once again, we assembled the parts through Golden Gate Cloning. We assembled the pBSGGIC plasmid with glpQ, phoD and PmaeN. Before testing the circuit, we built two CRISPR plasmids for the deletion of the native glpQ and phoD genes, creating the ΔphoD/ΔglpQ strain, . In this way, our strain should not release phosphate under low phosphate conditions as the system naturally would do. This means that we could then test the strain under low phosphate conditions without phosphate being released, which would interfere with the analysis of our results. After deletion of both genes, the circuit was transformed into B. subtilis.
We TESTED the strain by growing the cells in LB medium to OD600 = 0.8. Cells were spun down and washed to remove residual phosphate. Cells were then resuspended in malate supplemented solution for 1 hour, after which the supernatant was collected, and phosphate levels were measured using the commercial phosphate measuring kit (SpectroQuant®) (Fig. 7).
Figure. 7 Testing phosphate release in the presence of malate. B. subtilis cells, including the B. subtilis ΔphoD/ΔglpQ strain (no circuit) and cells with the integrated release circuit were grown (1 hr, 37 °C) without (circuit -ve) and with (circuit +ve) malate induction (1 mM) following resuspension of cells in 0.85% saline. Saline was included as a control. The supernatants from these cells were filter sterilised and tested using a commercial kit (SpectroQuant® Phosphate Test Cell) for phosphate release. Data are presented are the values from three independent biological replicates.
We LEARNT that B. subtilis ΔphoD/ΔglpQ circuit containing strain had a significantly higher release of phosphate upon malate induction. This allowed us to conclude that the circuit worked in the intended manner. This shows that we successfully went through the DBTL cycle, showing the success of our parts step by step leading to the understanding of how our final circuits worked.