Team:NAWI-Graz/Engineering
Team:NAWI-Graz
Engineering
After we decided to tackle the phosphate problem, we thought of ways to build a system that would be easy to deploy and that utilises bacteria that are already present in agricultural soil. Our approach included making phosphate more available to plants. For this purpose we came up with an approach to use the quorum sensing system of the rhizosphere inhabiting Bacteria.
BBa_K3928000
Design:
For the realization of our vision we needed to adapt our plasmid construct to an already existing system in E. coli. The phoR - phosphate regulon sensor protein is a membrane-associated protein that phosphorylates and thus activates phoB-proteins in response to limited environmental phosphate concentrations. As part of phoB-activation, a multitude of regulative operons associated to the phosphate metabolic pathway also get activated, among them the alkaline phosphatase promoter PphoA.
The reason we used this promoter specifically is that previous IGEM-competitors characterized its usage adequately for us to modify it to the degree we hoped for. In the course of our initial research period our original planned implementation for the negative regulation of PphoA in response to environmental phosphate concentrations posed a substantial problem: since the promoter activates at low and deactivates at high phosphate levels our system would not be able to fulfill its intended use of operating as soon as fertilizer (which contains phosphorus) is added. So we had to design a system.
Build:
Our goal was it to built a plasmid for E.coli detection by a system in which PhoB, which is native to E. coli binds to the used PphoA promoter (BBa_K1139200) activating transcription of iLacI (BBa_K3257045) which can bind to the LacI/LacO binding site (BBA_K3257066). By that, it prevents the expression of eGFP (BBa_K1911005) which is regulated via the constitutive promoter (BBa_J23106).
At low phosphate concentrations, the PphoA promoter is active, the LacI protein is expressed, which leads to a repression of the constitutive promoter via the LacO operating site. Thus no GFP is expressed.
At high phosphate concentrations, the PphoA promoter is inactive, the LacI protein does not get expressed, therefore GFP can be expressed due to the usage of a constitutive promoter.
We used the iGEM plasmid BBa_J04450 as the vector for our first attempt. E. coli Top 10 cells were transformed with our plasmid utilising the RFP expression of this plasmid as a marker for a successful transformation. After the transformation was completed, colonies were visible. The vector was extracted via miniprep.
The two insert parts, one with the PhoA promoter (BBa_K1139200), the LacI (BBa_K3257045) gene and the LacL/LacO binding site (BBA_K3257066), and the second one with the eGFP (BBa_K1911005) marker and a constitutive promoter (BBa_J23106) were synthesised. To have enough DNA material the two parts got separately amplified by PCR.
The construction of the BBa_K3928000 plasmid was done by restriction digestion and ligation. The PCR product of part 1 was digested with EcoRI and BamHI and the product of part 2 was digested with BamHI and PstI. The vector was digested with EcoRI and PstI. After this we checked the digestion of the parts by gel electrophoresis.
The bands of interest containing the digested parts were cut out of the gel and extracted. After checking for the desired parts, we ligated the 2 fragments described above as part 1 and part 2 with the vector.
The result was plasmid BBa_K3928000
Test
To assess whether our construct was functional we decided to do an assay as previous iGEM teams suggested [1]. As previously mentioned under the presence of phosphate our construct BBa_K3928000 should express GFP. Further we used various different concentrations of K2HPO4 as our phosphate source, to determine a signal threshold for excitation.
We started with an ONC of our E. coli TOP10 cells with BBa_K3928000 in MOPS minimal media [2] with phosphate (0.132 M K2HPO4) and chloramphenicol (25µg/ml) at 37°C. After diluting the cells to an OD600 of 0.1 in fresh MOPS media we incubated them further. Then the cells were centrifuged and washed twice with fresh MOPS without phosphate and resuspended to an OD600 of 10.
To determine the concentration necessary for eGFP excitation, 300µl of cells were incubated at 26°C with different concentrations (4µM, 5µM, 10µM, 50µM, 100µM, 1000µM) of K2HPO4 in MOPS media with chloramphenicol (25µg/ml). After 120 minutes, fluorescence intensity was measured.
Learn:
The Phosphate Assay did not provide a valid result. Therefore we were not able to confirm the functioning of our part (BBa_K3928000).
The photon emission of the expressed GFP should have been measured in a fluorimeter. After stimulation with light at the excitation wavelength, no emission at the target wavelength could be measured. We could not assess what the reason for that was. It is possible that the plasmid for the expression of the GFP was not inserted into the cells properly with the transformation. The phosphate concentrations in the assay could have been to low, or the plasmid was not abundant in the cells in high enough numbers to produce enough GFP for a measurement.
BBa_3928003
Design:
This plasmid was created with the basis of the BBa_K3928000 plasmid, by exchanging the eGFP of the positive-phosphate-phoA-GFP-sensor (BBa_3928002) by the autoinducer synthetase for AHL (BBa_C0061)
This, then, should lead to the excretion of AHLs as quorum sensing molecules. The cell containing BBa_3928003 thereby works as a sender cell that reacts to phosphate akkumulation. The receiver cell should react to these AHLs by expressing marker genes like GFP.
At low phosphate concentrations, the PphoA promoter is active, the LacI protein is expressed, which leads to a repression of the constitutive promoter via LacO and no expression of the AHL synthase. Thus no quorum sensing molecule 3OC6HSL gets produced. In this scenario, the receiver cell perceives no quorum sensing molecules and should not react in any way.
At high phosphate concentrations, the PphoA promoter is inactive, the LacI protein does not get expressed.This leads, as in BBa_K3928000 described, to the expression of the AHL synthetase which produces quorum sensing molecules (3OC6HSL). This should then be detected by the receiver cells, which should express a marker molecule in response to the quorum sensing molecules.
Build:
To generate BBa_3928003, we tried to use the Gibson cloning method.
Two sets of primers were designed for the gibson assembly. The first one amplified the LuxI gene of interest with the additional assembly overhangs.
The second one amplified the vector constructed from BBa_K3928000 but without eGFP.
If properly assembled via gibson assembly, the created plasmid then should be brought into E.coli via transformation. The resulting transformed cell should then excrete the quorum sensing molecules described above.
Unfortunately the gibson assembly was not successful which denied us from going on further in our research.
References
[1] Marie-Andre´e Dollard, Patrick Billard; Whole-cell bacterial sensors for the monitoring of phosphate bioavailability, 2013
[2]Frederick C. Neidhardt, Philip L. Bloch, David F. Smith;
Culture Medium for Enterobacteria, 1974