The goal of iGEM24-SCUT-China-A is to use synthetic biology tools to obtain Halomonas TD strains that can tolerate acetate and metabolize formate, build a thermalsensitive bio-switch suitable for it and subsequently apply them to the production of P34HB, tyrian purple, proteins.

Firstly, we conducted adaptive laboratory evolution on Halomonas TD01 to improve its ability to utilize acetate, the main product of CDE. After domesticating the TD80 strain, we chose to introduce the formate assimilation pathway to enable it to utilize formate, a difficult-to-recover product in CDE.
For the first method, based on previous studies obtained from literature research, we selected the tetrahydrofolate (THF) cycle imported from Methylobacterium extorquens AM1.
As a second approach, based on the homology between Vibrio natriegens and Halomonas TD, we chose to import the C1 modules from Vibrio natriegens.
After optimizing the efficiency and tolerance of Halomonas TD in utilizing carbon dioxide derivatives, we began to explore its production pathway and potential, resulting in the production of P34HB, tyrian purple, indigoidine, proteins.

We divided our thermal bio-switch into two parts, which are carried by pSEVA321 and pSEVA341 respectively. After successfully constructing the T-switch suitable for Halmonas TD, in order to further optimise its performance, we replaced the promoter of the regulatory protein and the reporting gene respectively,and mutated the regulatory protein. After five months of experiments, we obtained a certain range of various versions of T-switches with different expression intensity and we decided to use the them to compete for the Best Part Collection awards.

We introduced the porin194-4hbd-sucD-ogdA-porin194-orfZ gene cluster using pSEVA321 and pSEVA341, respectively. We found that the 4HB molar ratio obtained by introducing pSEVA321 was higher than that of pSEVA341, but the dry weight decreased to some extent compared to the wild TD80. In order to stabilize cell dry weight while increasing the 4HB molar ratio, we decided to change the screening pressure and knock out the cysNC gene encoding a key enzyme in the sulfate assimilation pathway in Halomonas TD, in order to block the sulfur source supply for methionine synthesis. At the same time, cysNC was added to pSEVA321 backbone to screen for strains that had been introduced with the 321-porin194-4hbd-sucD-ogdA-porin194-orfZ plasmid

Since two key enzymes( SttH and TnaA) would compete with each other for the tryptophan substrate ,we introduced T-switch into the biosynthesis pathway of Tyrian purple using parts below.

To assess the capability of Halomonas TD to produce PhaP and SOD proteins, we selected the Mmp1 inducible promoter for protein expression.