Part:BBa_K2055369

Na(+)/H(+) antiporter NhaA generator

This construct codes for intermembrane protein Na(+)/H(+) antiporter NhaA, which is a proton pump from E. coli capable of excreting one intracellular Na(+) ion in exchange for two H(+) external protons. This protein is active at alkaline pH. This construct is regulated by a weak constitutive promoter and its objective is to augment bacterial resistance to alkaline pH.

We transformed E. coli BL21 with three different constructs, one coding for the proton pump, and the other two were other constructs that do not code for an alkaline resistance that were on the same vector so that they had the same antibiotic resistance, ampicilin. We compared the growth rate at a constant alkaline pH of 9.3, that was manually adjusted with KOH, at a growth temperature of 37ºC and 250 rpm. We conducted our experiments in triplicate, measuring the OD600 of each of them in approximate intervals of 60 minutes for 8 hours. We calculated an average of the three replicates for each transformed strain and graphed the data in order to determine if the proton pump had a significant effect on the survivability of the bacteria. The strain transformed with the proton pump had an exponential growth, while the others couldn’t resist the alkaline pH and had minimal growth. This modification to bacterial cell membrane may increase stress resistance under alkaline conditions.

(by maintaining the pH within the cell at a constant level, this is done by the entrance of two protons and exit of a sodium cation.)

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 415
  Illegal BamHI site found at 1120
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 382
  
• 1000
  COMPATIBLE WITH RFC[1000]