Coding

Part:BBa_K3595008

Designed by: Ying Li   Group: iGEM20_GZ_HFI   (2020-10-26)


Mutant cysE-5-11-2

cysE-5-11-2 is a mutant of cysE gene, which is obtained by combining the positions of mutations in mutants cysE-5, and cysE-11-2.In the pathway of converting hydrogen sulfide to L-cysteine, L-serine and Acetyl-CoA form O-acetylserine catalyzed by L-serine O-acetyltransferase (SAT). SAT is encoded by gene cysE, and its feedback is inhibited by the final product L-cysteine . In order to induce overproduction of L-cysteine, we need to develop a mutant gene of cysE which will code for feedback inhibition-insensitive SAT.We tried to construct different mutants through site mutation, and cysE-5-11-2 is one of the different mutants.

Usage and Biology

This part can be used as a coding sequence after the promoter pTac and RBS B0034. The feedback inhibition-insensitive SAT can be translated under the induction of IPTG. We constructed plasmids pBR322-KanR-pTac-cysE and pBR322-KanR-pTac-cysE-Mutant,among which the mutants include cysE-256, cysE-5, cysE-11-2, cysE-5-11-2, cysE-256-5,cysE-256-11-2,cysE-256-5-11-2. The constructed plasmid was transformed into Nissle host cell to test its production of cysteine.

The structure of the plasmid pBR322-KanR-pTac-cysE and pBR322-KanR-pTac-cysE-mutant

Experimental Setup

  • Genetic information of cysE,cysE-256, cysE-5, cysE-11-2, cysE-5-11-2, cysE-256-5,cysE-256-11-2,cysE-256-5-11-2 was described on the page ofPart:BBa_K3595004, Part:BBa_K3595005,Part:BBa_K3595006,Part:BBa_K3595007,Part:BBa_K3595009, Part:BBa_K35950010, Part:BBa_K3595011,respectively.
  • Plasmid pBR322-KanR-pTac-cysE and pBR322-KanR-pTac-cysE-mutant was transfered into the Nissle host cell,respestively.
  • Single colonies were selected from the experimental LB-agar plate , then inoculated into test-tube tubes with 4000 μL LB liquid medium with 4uL kanamycin for overnight growth at 37 °C and 200 rpm.
  • Inoculating 15 uL of culture solution overnight into a 24-well plate containing 3 mL M9 medium for overnight growth at 37 °C and 200 rpm.The media contained 3 ul kanamycin and 1.5 uL 1M IPTG. At the same time, wild-type Nissle was inoculated as negative control, and M9 medium was used as blank control.
  • Detecting cysteine concentration in culture medium

Results

  • All of our engineered bacteria have shown great improvement in the ability to produce cysteine, which represents that they can absorb H2S to a larger extent.
  • EcN with plasmid pTYT-cysE-5-11-2 had the best effect, 98.72% batter than EcN wildtype and 8.79% better than overexpression of cysE (pTYT-cysE)
Detection results of the effect of the hydrogen sulfide pathway. (A) The calibration line of cysteine. (B) Concentration of cysteine detected in each group. (C) The ratio of increased cysteine concentration in each group compared to wildtype. (D) Cysteine production compared to pTYT-cysE. CysE-mut in the Figure stands for cysE-256.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 684
  • 1000
    COMPATIBLE WITH RFC[1000]

Reference

Kai Y, et al. Engineering of Escherichia coli L-serine O-acetyltransferase on the basis of crystal structure: desensitization to feedback inhibition by L-cysteine. Protein Eng Des Sel 2006;19(4):163-7.


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