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
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 969
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000Illegal BsaI site found at 789
Illegal BsaI.rc site found at 1181
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.
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).
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.