There is a problem when we try to confirm the function of Pst system that we transferred can achieve our goals. The wild-type E.coli has an inherent regulation mechanism, which means the Pst system will be repressed if the concentration of phosphate is higher than 1mM and it is much more difficult to find and knockout the regulator genes preciously. But we find that the mainly negative feedback regulation is through the combination between phoB protein and Upstream promoter of the PstS gene. Therefore, the Pst gene we transferred will not be controlled by this way. However, unavoidably, there may be other unknown negative feedback regulations, that is, the overexpression of Pst could minimize the effect caused by them.
Firstly, we try to find a proper concentration of inducer to against the negative regulation. We set different levels of inducer (IPTG) and finally detect the protein content in the cell. We have found that 2mM IPTG is enough to observe the target protein in 4 hours. Then we used 2mM IPTG to induce the expression of Pst system under different time conditions, and the result shows that the difference between the two groups can be maximum when it reaches 6 hours and it seems that the increase in the difference has been hard to observe as time goes on.

This part focuses on the construction of a PPK mutant element that can significantly enhances the ability of E. coli to convert Pi to PolyP for storage in vivo.
The ability of our modified mutant to store phosphorus is characterized indirectly by measuring the difference in phosphorus concentration within the bacterial medium at the same growth density of the wild-type and mutant strains. The specific steps on how to obtain and modify wild-type ppk gene can be found in the wet lab_experiments section.

DNA fragments containing yjbB was amplified from E. coli MG1655 genomic DNA using the primers yjbB-5’/yjbB-3’.The PCR fragments were inserted into the Xbal/Xhol sites of pET-22b(+).

Two plasmids, cI857-PR-phlf-mCherry plasmid and Pphlf-eGFP plasmid, were transferred into BL21 strain. The mechanism of temperature regulation of cI857 protein structure has been introduced in the cI857 element section. We use different temperatures to culture the strain. It is estimated that cI857 forms dimer under 30℃ culture conditions to inhibit the R promoter, so that its downstream phlf and mCherry do not express. The phlf promoter in another plasmid can normally turn on the expression of green fluorescent protein eGFP. At this time, green fluorescence can be detected without red fluorescence using the microplate analyzer; The cI857 dimer was depolymerized at 37℃, and the inhibition of the R promoter was relieved. The downstream phlf and mCherry were normally expressed, and phlf inhibited the phlf promoter in another plasmid, so that the green fluorescent protein eGFP was not expressed, while mCherry expressed red fluorescence. At this time, red fluorescence should be detected instead of green fluorescence.