Design
In E. coli, there naturally exists a pathway that the organism uses to fine-tune the concentration of manganese within the cell that revolves around the Manganese Transport Regulator (MntR) protein. When designing our gene block, we used the mntP (exporter) promoter and riboswitch in this pathway that respond to manganese. As a response to the manganese, we made our gene block produce superfolder GFP (sfGFP) as a simple reporter. Finally, we put a BBa_B0015 terminator at the end of our gene block. This is a “commonly used” and “reliable” double terminator (B0010-B0012) from the iGEM registry.
We also built another construct to control expression of MntR. The lac operon in our backbone is inducible by IPTG, allowing precise control over how much mntR is generated by the cell. This could help us control the sensitivity of our sensor. The MntR produced by this construct will contain a 6xHIS tag to aid in back-end testing, such as in immunoblotting.
Build
We had a pTrc backbone with an insert transformed into bacteria from last year’s project. This plasmid contained cut sites for NcoI and HindIII surrounding the gene block. These enzymes were used to excise the old gene block and leave us with linearized pTrc backbone. Using HiFi cloning and the 20bp regions of homology between our gene block and our backbone, we cloned the 6xHIS-mntR overexpression gene block into the plasmid and transformed the DNA into MG1655 WT E. coli.
In addition, last year, we received dried pSB3K3-RFP plasmid in a distribution from iGEM. This dried plasmid was resuspended in water and transformed into NEB5α cells. EcoRI and SpeI were used to cut pSB3K3 and remove the RFP gene. We used HiFi Assembly to insert our sensor gene block (pmntP-rs-sfGFP), received from IDT, into pSB3K3. We used a New England Biolabs kit to do the assembly as well as the subsequent transformation into provided MG1655 WT E. coli cells.
Test
Our initial manganese screening involved a dilution series of manganese (II) chloride0.001mM – 110mM. Borrowing a protocol from the Air Force Research Laboratory, we grew cells at 37°C at 250rpm for 2 hours toan A 600 of 0.5. Manganese chloride solution was added to the cultures in a log dose series of concentrations from 0.001mM – 110mM. A treatment similar to that of the MG1655 cells was performed on a positive GFP control (pET29b-eGFP) with 1mM IPTG to induce transcription. Cultures of 1.5mL were treated with 150uL of 1M IPTG and left in incubation for 3 hours. While the fluorescence was not visible by pelleting the bacteria and examining on a UV table, we were able to measure fluorescence using our BioTek Synergy H1 plate reader. The dose response curve showed that our pSB3K3-pmntP-rs-sfGFP sensor in wild-type MG1655 E.coli produced sfGFP in response to manganese chloride in the low mM range (1-10mM). The higher tested doses (not shown) inhibited the growth of E.coli. The plasmid was also transformed into MG1655 cells with MntR deleted, a strain in the E.coli Keio collection obtained from Horizon Discovery (part of Perkin Elmer) and generated by Baba et al 2006. When treated with manganese, these cells did not produce fluorescence seen in wild type cells, indicating that the sensor was functioning in a manner dependent on the MntR pathway, and thus specific to manganese levels.
APA Cite
Baba, T., Ara, T., Hasegawa, M., Takai, Y., Okumura, Y., Baba, M., Datsenko, K. A., Tomita, M., Wanner, B. L., & Mori, H. (2006). Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Molecular systems biology, 2, 2006.0008. https://doi.org/10.1038/msb4100050
Learn
Our sensor has an active range between 0.1-10mM. In subsequent tests, we will test additional concentrations in this range. By doing this, we will both flesh out the dose-response curve as well as find what concentration of manganese becomes lethal to the cells. In addition, we had large amounts of background fluorescence in our readings. LB media is made with yeast extract, so we suspect it inherently contains manganese. An alternative media that we could try is M9, a minimal salts media with no manganese. Finally, our sensor is not sensitive enough to detect low, dangerous levels of manganese. The upper limit for safe manganese levels is 0.5ppm (O'Neal), which is equal to 0.009 mM. Our sensor only goes down to 0.1mM. We will try to use pTrc-mntR to control the amount of MntR in the cell and improve the sensitivity of the sensor.Sources:
BBa_B0015 Link: https://parts.igem.org/Part:BBa_B0015
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4545267/
O'Neal SL, Zheng W. Manganese Toxicity Upon Overexposure: a Decade in Review. Curr Environ Health Rep. 2015 Sep;2(3):315-28. doi: 10.1007/s40572-015-0056-x. PMID: 26231508; PMCID: PMC4545267.