Part:BBa_K2232012

P43_OF4-nhaC_rrnBT1

The gene nhaC from the Bacillus pseudofirmus OF4 encodes the Na+/H+ antiporter, which regulates cytoplasmic pH value by coupling net H+ uptake with Na+ extrusion. NhaC, the first demonstrated alkaliphile Na+/H+ antiporter, as part of a complement of antiporters in the wild-type alkaliphile, apparently plays a role over a wide range of pH values. Its most apparent role was observed at pH 7.5 in media containing low Na+ concentrations, where a pronounced defect in growth was observed. Although pH homeostasis is not a major challenge at pH 7.5, the alkaliphile must nonetheless extrude Na+ that enters in symport with solutes, including the growth substrate malate in the media used here. At pH 10.5, NhaC also plays an apparent role at low Na+ concentrations;at the alkaline pH, it contributes to pH homeostasis and may also contribute to the capacity of the cell to lower the cytoplasmic Na+ concentration optimally.

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 NgoMIV site found at 527
  
• 1000
  COMPATIBLE WITH RFC[1000]

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 OF4-nhaC(Fig.1) ,which plays a role as shelter protecting B.subtilis from Alkaline environment.

Fig.1 Construction of the expression vector_P43-nhaC-T1. The P43 and T1 represent strong promoter P43 from B.subtilis and terminator T1 from E. coli rrnB. The part OF4-nhaC was inserted by the restriction site KpanI at 4430 bp and HindIII at 5194 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 733 bp and 6703 bp respectively, which correspond to the length of OF4-nhaC and the blank plasmid. Lane 1: Complete plasmid; Lane 2: Plasmid digested by KpnI and HindIII; Lane M: DL marker.

After transformation of this part, The recombinant B.subtilis WB800_nhaC was grown in LB culture for 24h and the cells were disrupted by sonication in 20 mM Tris-HCl(PH 8.0) buffer. The lysate was then centrifuged and the precipitate were electrophoresed on a sodium dodecyl sulfate(SDS)-12% (wt/vol) polyacrylamide gel, followed by Coomassie blue staining(Fig.3).

Fig.3 SDS-PAGE analysis of membrane protein of original B.subtilis and the transformant of OF4-nhaC. Lane M: Marker ladder; Lane 1 & 2: The recombinant strain WB800_nhaC; Lane 3: Original strain WB800. Lane 1 & 2 showed the same band(in red box) corresponded with the molecular weight of NhaC(36kDa).