Team:BIT-China/Experiments

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  Description Design Basic circuits Fine-regulation circuits
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Error-prone PCR

Similar with Polymerase chain reaction (PCR), error-prone PCR is also a powerful method to produce linear DNA fragments, but results in random mutagenesis. This mutagenic PCR, which introduces random mutations by reducing the fidelity of the DNA polymerase. This process is similar to standard PCR cloning of a target sequence except that, for constructing a diverse mutagenesis library, a much larger pool of transformants are needed to increase the diversity of the library, making the process tedious and inefficient. As a result, various processes have been developed to streamline the methods.

The process of mutating P-atp2 is cockamamie, when it does not require excellent skill of experiment or advanced technology. Even though there are several ways to change the reaction condition, we finally chose to put manganese ion into our error-prone reaction system. So our error-prone PCR was performed in 25 μL reaction solution (Table.1).

Table.1 the reaction solution we used to get the P-atp2 mutational fragments

10x buffer	2.5 μL
primer	4.0 μL
template of P-atp2 fragment	1.0 μL
dNTP for error-prone	2.5 μL
magnesium ion(Mg2+) solution	2.0 μL
rTaq DNA polymerase	0.5 μL
manganese ion(Mn2+) solution	0.125μL, 0.25 μL, 0.375 μL, 0.5 μL
water	Added to 25μL

Measurement of enzyme activity

To verify the promoters after the error-prone PCR, we used LacZ alpha, encoding the enzyme β-galactosidase with LacZ beta, as a report gene to reflect the ability of promoters. With the purpose of high throughput screening promoters, we measured them on the 96-well cell plate. As these bacteria grew normally and the OD₆₀₀ was up to 1.5, we lysed cells, then added ONPG to start a color reaction and recorded the response time(t). After the sodium carbonate was added to terminate the reaction, we measured the OD₄₂₀ and OD₅₅₀, which could be put into the formula to calculate the activity (Table.2).

Table. 2(A). The system of each 96-well

culture	LB	30 μL
lysing	Z-buffer(2-Mercaptoethano)	180 μL
	isopropanol	20 μL
	0.1%SDS	10 μL
color reaction	ONPG	40 μL
ending the reaction	Na2CO3	60 μL

Table. 2(B). The formula to calculate the activity of β-galactosidase

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

One-step Site-directed Mutagenesis

We inserted target sequence into plasmid to construct our alkali induced promoter P-atp2. However, there is an EcoRI site exists in its sequence. So we must change it for standardizing it. Our protocol is rather simple. We just use PCR to perform it, one-step site-directed mutagenesis. In our whole experimental processes, we used it many times to the EcoRI site mutation.