Latest revision as of 14:10, 12 October 2022

polyP sensor without BBa_M0052 SsrA degradation tag

Usage and Biology

Design

Concluding from the previous experiments, AsPhoU cell has proven its effectiveness in absorbing a higher amount of Pi in the environment. While acknowledging that, we simultaneously have to make sure that the absorbed Pi constructively fixates into inorganic polyphosphate (polyP) so that it remains inside the bacterial bodies. Henceforth our team utilized Sigma-Aldrich’s PolyP assay kit, which helped in quantifying the amount of PolyP in bacterial bodies. The design of the polyP sensor plasmid includes genes encoding for mCherry fluorescent protein and RpoD sigma factor with a P region that easily binds to polyphosphate, respectively. Without polyphosphate accumulation, the sigma factor could successfully direct the RNA polymerase to the promoter, resulting in the expression of mCherry fluorescent protein. However, if polyphosphate is fixated, accumulated, and attached to the P region of the sigma factor, the sigma factor loses its function and would be unable to direct the RNA polymerase for transcription of mCherry. Therefore, the increase in phosphate absorption, which increases phosphate fixation, results in reduced mCherry fluorescence. By measuring the mCherry fluorescence, we could monitor the accumulation of polyP in the cell. The method is employed in the design of our implementation. To ensure that our AsPhoU cells perform at their highest efficiency, we designed the hardware to detect reduced fluorescence levels to the minimum, which signals that the cell has reached maximum phosphate fixation. A notification would then be sent through the designed software to remind the users to replace the filter in time, thus maintaining the effectiveness of our device and further ensuring biosafety. For further information on the implementation design, please visit our Proposed Implementation page.

Build

We committed to Twist Bioscience (ABreal Biotech Co., Taiwan) for synthetic ropD gene ((BBa_K4271010)), comprising N-terminal 69 amino acid for polyP binding (Yang et al., 2010), E. coli RpoD (1-555 a.a), C-terminal 4.2 region for UreA promoter recognition (BBa_K4271011) (Beier et al., 1998). We conducted reverse PCR to amplify the plasmids, consisting of mCherry (BBa_J18932), and transcriptional terminators (BBa_B0015). Two fragments, Tac promoter (BBa_K4271009) and transcriptional terminators (BBa_B0015) were prepared by PCR. We also amplified the genetic synthetic UreA promoter (containing RBS, BBa_K4271012) by PCR, respectively. Due to time constraints, we weren’t able to complete the engineering cycle of this particular construct. Yet we have drawn up a future plan for the building of the polyP sensor. Tac promoter (189 bps), synthetic ropD gene (1986 bps), transcriptional terminator (173 bps), and plasmid (2885 bps) will be connected by Gibson assembly to generate the polyP sensor (Fig. 14). The assembly product will then be transformed to E. coli DH5 alpha via the heat shock method. Colony PCR with two primers, PTac _forward and Tt_reverse, will be used to confirm and generate a 3.7 bps fragment if the assembly succeeded.

Future Plans

Owing to time constraints, we have not been able to test the ability of AsPhoU for intracellular PolyP accumulation. Regardless, we would conduct this experimental process in the future, with the hope of reiterating that our proposed solution successfully operates. Moreover, we would subclone oph and AsPhoU to pACYCDuet vector with p15A ori, compatible with PolyP sensor carrying pMB ori to generate our PolyP sensor cell in this project.

References

Yang, Z. X., Zhou, Y. N., Yang, Y., & Jin, D. J. (2010, June 11). Polyphosphate binds to the principal sigma factor of RNA polymerase during starvation response in Helicobacter pylori. Molecular Microbiology, 77(3), 618–627. https://doi.org/10.1111/j.1365-2958.2010.07233.x

Beier, D., Spohn, G., Rappuoli, R., & Scarlato, V. (1998, October). Functional analysis of theHelicobacter pyloriprincipal sigma subunit of RNA polymerase reveals that the spacer region is important for efficient transcription. Molecular Microbiology, 30(1), 121–134. https://doi.org/10.1046/j.1365-2958.1998.01043.x

Sequence and Features