Part:BBa_K1675021

P-atp2 mutant 705-B0034-lacz alpha

The natural P-atp2 is an alkali-induced promoter in Corynebacterium glutamicum located in F0F1 ATPase operon. The natural P-atp2 promoter responds to the pH changes from pH 7.0 to pH 9.0, especially at alkaline pH. When the pH value increasing, the expression increased modestly. It is activated by the alternative sigma factor of the RNA polymerase, whose synthesis would be activated when the bacteria are growing at alkaline pH.

The activity of this mutant at different pH is shown in figure 1.

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The verification of alkali-induced promoter P-atp2's transcription function (BBa_K1675021) Data

At first,we used error-prone PCR to mutate the natural P-atp2 and get various P-atp2 mutants, which have different efficiency and different respongsing ranges. The steps of EP-PCR are shown below (Fig.1).

Fig.1 The four steps of EP-PCR.

Then, we mutated the EcoRI site of the promoter by single-step mutation. And we determined the activity of P-atp2 in different pH from 5.0 to 9.0 by measuring the activity of enzyme β-galactosidase(LacZ). As these bacteria grew normally and the OD₆₀₀ was up to about 1.5, we lysed cells, then added ONPG to start a color reaction and recorded the response time. After adding the sodium carbonate solution (1M) to terminate the reaction, we measured the OD₄₂₀ and OD₅₅₀, which could be put into the formula to calculate the activity(Table. 1).

Enzyme Activity= 1000*(OD₄₂₀-1.75*OD₅₅₀)/(t*0.1*OD₆₀₀)

Table. 1 The formula to calculate the enzyme activity of β-galactosidase. According to the β-galactosidase activities (Fig.2), we can get the transcription ability of P-atp2 under the different pH environment.

Fig.2 The β-galactosidase activities in different pH from 5.0 to 9.0.

Austin_UTexas 2016 iGEM Team Characterization

Characterized by Austin UTexas in 2016

The above part served as the promoter in conjunction with the BioBrick BBa_K592009 which served as the visual reporter for the design of a pH sensor for use in E. coli. A test was designed in which a plasmid containing the P-atp2 promoter with the blue chromoprotein was grown alongside an E. coli line that contained a plasmid with the blue chromoprotein and the promoter/RBS BBa_K806002 (BBa_K209701). We expected to see constant blue chromoprotein production in the control series (those that lacked P-atp2) and a gradual visual increase in blue chromoprotein as the pH was raised from 6 to 9 in the cells that contained the P-atp2 construct.

Figure 1

As seen in figure 1, there is no clear gradual increase in blue chromoprotein in the P-atp2 trials suggesting that this promoter is not sensitive to basic pH changes. Spectrophotometer readings were unable to be taken due to the blue chromoprotein's absorption at 588nm interfering with the 600nm wavelength used to take cell counts, meaning quantitative data was not discernible between what was cell, and what was blue chromoprotein.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]