Difference between revisions of "Part:BBa K3733003"

Revision as of 18:08, 20 October 2021

ThsS(L547T)：A point mutation of ThsS

ThsS(L547T）is improved from the ThsS(https://parts.igem.org/Part:BBa_K2507000), which is the membrane-bound sensor kinase (SK) which can sense thiosulfate outside the cell.

usage and Biology

ThsS (BBa_K2507000) and ThsR (BBa_K2507001) are the two basic parts of the two-component system of the marine Shewanella species. When the thiosulfate outside the membrane is felt, ThsS will be phosphorylated, and then it will phosphorylate ThsR. In turn, the phosphorylated ThsR can activate the PphsA promoter to control the expression of downstream genes. ThsS(L547T) is a mutant of ThsS, which amino acid at position 547 is mutated from leucine to threonine, which can lower the threshold of ThsS to sense thiosulfate theoretically. At the same time, this mutation also makes the expression of downstream gene under the control of ThsS (L547T) and ThsR two-component system.

Functional Parameters

To characterize this part, ThsS and ThsS(L547T) were cloned into pSC101 vector separately. We chose neGFP(https://parts.igem.org/Part:BBa_K3733012) as the reporter. Plasmids were transferred into E. coli DH5α. The strain was expanded in LB medium to OD=0.4, then 198μL of bacterial solution was spotted into a 96-well plate, and a series of concentration gradients (0mM, 0.001mM, 0.01mM, 0.1mM, 1mM, 10mM) of thiosulfate were added Sodium sulfate solution. Add 2μL of sodium thiosulfate to each well for induction and measure the fluorescence with gain of 50 and OD₆₀₀ in the Synergy H1 microplate reader overnight. Since we have successfully achieved an order of magnitude change in NarX(https://parts.igem.org/Part:BBa_K3733000) sensing threshold by modifying the combination of the promoter and RBS controlling the NarXL two-component system, we did not set a more detailed concentration gradient in this experiment. However, through the data obtained from our experiments, we found that the mutated ThsS can increase the expression level of downstream gene (Figure 1).

Sequence and Features

Reference

Landry BP, Palanki R, Dyulgyarov N, Hartsough LA, Tabor JJ. Phosphatase activity tunes two-component system sensor detection threshold. Nat Commun. 2018 Apr 12;9(1):1433.