Part:BBa_K4644004

pgsBCAE

Bacillus subtilis strain A-5. pgaBCAE protein coding region.This part is useful for y-PGA synthesis.(The part included in the ellipse is the Polyglutamate synthetase catalytic part）

Fig 1.Degradation of urea

Biology

        In B. subtilis pgsBCA and the required factor (pgsE) were essential for y-PGA synthesis , which functioned as a synthase complex. The transmembrane and subcellular localization of related proteins were predicted by TMHMM and PSORTb.

        pgsB possessed a full length of 1182 bp nucleotides encoding 393 bp of amino acids , which could catalyze y-PGAsynthesis intracellularly as a non- transmembrane protein by anchoring to the plasma membrane through an anchor hook.

        pgsC contained 450 bp nucleotides encoding 149 aa; pgsC was predicated to have four transmembrane helixes and to be fixed on the plasma membrane to connect pgsB and pgsA.

        Protein pgsA was predicted to contain one transmembrane helix in the 380 aa and to be anchored to the plasma membrane through the N- terminal transmembrane region and anchor hooks. Therefore, pgsA is also responsible for the extracellular transport of y-PGA.

        pgsE was annotated to encode the factor required for y-PGA synthesis and one transmembrane helix was predicted in the membrane with 27-55 aa.

usage

        We ligased the strong promoter and RBS and the parts (pgsBCAE) on the expression vector PETlac by standard assembly. Then the ligation mixture was transformed into E. coli nissle 1917 & E. coli BL21(DE3), which enabled the E. coli to express pgsBCAE protein.

Fig 2. PETlac-pgsBCAE

Characterization

        The pgs promoter from Bacillus subtilis strain A-5 plays a crucial role in the synthesis of polyglutamic acid. It utilizes racemic glutamic acid as a raw material to synthesize polyglutamic acid and then expels the product extracellularly. We obtained it through PCR from the genome of Bacillus subtilis strain A-5.

Fig 3. The result of pgsBCAE

        After attaining the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure 4.

Fig 4. The result of plasmid cut with enzyme KpnI(F) and BamHI(R). Plasmid: PETlac.

Reference

Bai, N.; He, Y.; Zhang, H.; Zheng, X.; Zeng, R.; Li, Y.; Li, S.; Lv, W. γ-Polyglutamic Acid Production, Biocontrol, and Stress Tolerance: Multifunction of Bacillus subtilis A-5 and the Complete Genome Analysis. Int. J. Environ. Res. Public Health 2022, 19, 7630.

Xiaoyu Wei, Zhen Chen, Ailing Liu, Lijie Yang, Yiyuan Xu, Mingfeng Cao, Ning He,Advanced strategies for metabolic engineering of Bacillus to produce extracellular polymeric substances,Biotechnology Advances,Volume 67,2023,108199,ISSN 0734-9750,

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 737
  Illegal XbaI site found at 1032
  Illegal PstI site found at 382
  Illegal PstI site found at 1013
  Illegal PstI site found at 2028
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 737
  Illegal PstI site found at 382
  Illegal PstI site found at 1013
  Illegal PstI site found at 2028
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 737
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 737
  Illegal XbaI site found at 1032
  Illegal PstI site found at 382
  Illegal PstI site found at 1013
  Illegal PstI site found at 2028
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 737
  Illegal XbaI site found at 1032
  Illegal PstI site found at 382
  Illegal PstI site found at 1013
  Illegal PstI site found at 2028
  Illegal NgoMIV site found at 1277
  Illegal AgeI site found at 2968
  
• 1000
  COMPATIBLE WITH RFC[1000]