Difference between revisions of "Part:BBa K1675018"

Latest revision as of 17:24, 18 September 2015

P-atp2 mutant 154-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 Fig.1.

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Usage and Biology

The verification of alkali-induced promoter P-atp2's transcription function (BBa_K1675018) 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 responsing ranges. The steps of EP-PCR are shown below (Fig.1).

Fig.1 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.

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]

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-At first,we used error-prone PCR to mutate the natural P-atp2 to get various P-atp2 mutants, which have different efficiency and different ranges of response to the environment pH. The steps of EP-PCR are shown below (Fig.1).
+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 responsing ranges. The steps of EP-PCR are shown below (Fig.1).
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-Then, we mutated the <i>EcoRI</i> 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 OD<sub>600</sub> was up to about 1.5, we lysed cells, then added ONPG to start a color reaction and recorded the response time. After the sodium carbonate solution (1M) was added to terminate the reaction, we measured the OD<sub>420</sub> and OD<sub>550</sub>, which could be put into the formula to calculate the activity(Table. 1).
+Then, we mutated the <i>EcoRI</i> 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 OD<sub>600</sub> 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 OD<sub>420</sub> and OD<sub>550</sub>, which could be put into the formula to calculate the activity (Table.1).
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