Part:BBa_K2232003

C125-TupA

This part is the coding sequence (CDS) of a cytoplasmic protein TupA (GenBank: BAB07375.1), which catalyzes the conversion of glucuronic acid and L-glutamic acid to polyglucuronic acid and poly-γ-L-glutamic acid.

Usage and Biology

Gene tupA was cloned from the chromosomal DNA of facultative alkaliphilic strain Bacillus lentus C-125. The primary translation product of this gene, TupA, is likely a cytoplasmic protein(57.3 kDa) consisting of 489 amino acid residues.It was demonstrated that TupA is involved in the synthesis of TUP,a copolymer of polyglutamic acid (PGlu) and polyglucuronic acid (PGlcU),which is one of major structural components in the cell wall of the Bacillus lentus C-125 and can neutralize the extracellular hydroxyl. A mutant defective in TUP synthesis grows slowly at alkaline pH,indicating this gene plays a key role in pH homeostasis and increases the alkali resistance of Bacteria.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 969
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 789
  Illegal BsaI.rc site found at 1181
  

iGEM2017 SZU-China

In order to strengthen the alkali tolerance of B.subtilis, which is essential for our chassis to live in the concrete, we constructed an expression vector containing part C125-TupA(Fig.1) ,which plays a role as shelter protecting B.subtilis from Alkaline environment.

Fig.1 Construction of the expression vector_P43-tupA-T1. The P43 and T1 represent strong promoter P43 from B.subtilis and terminator T1 from E. coli rrnB. The part C125-tupA was inserted by the restriction site KpanI at 4430 bp and HindIII at 6001 bp.

We transformed the expression vectors into Bacillus subtilis WB800 by optimization of Spizizen method, and the real positive clones was confirmed by kanamycin screening and nucleic acid electrophoresis (Fig.2).

Fig.2 1% Agarose Gel Electrophoresis of DNA extracted from the positive clones and its identification by restriction digestion. The product of plasmid digested showed two signal bands at 1540 bp and 6724 bp respectively, which correspond to the length of C125-tupA and the blank plasmid. Lane 1: Complete plasmid; Lane 2: Plasmid digested by KpnI and HindIII; Lane M: DL marker.

we used the alkaline solid medium containing Na2CO3 to plant the transformed strain WB800_tupA and the original strain WB800. After 48hours, the plates of same pH value were dyed with crystal violet followed by washing with ddH₂O and compared the size of bacteria ring(Fig.3), which indicates the function of tupA to increase the alkali tolerance of B.subtilis.

Fig.3 Growth situation of original and tramsformed strains at different pH. The bacteria rings of WB800_tupA are always bigger than original WB800, especially at pH 9, which indicated the function of gene tupA as respected.