A specific biosensor for pyruvate (pyr) and its application
A biosensor, pyruvate technology, applied in the field of genetic engineering, can solve the problems of cumbersome detection technology, limited screening efficiency, and inability to detect molecular concentration in time
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Embodiment 1
[0030] Embodiment 1: Construction of PYR sensor plasmid pYR02
[0031] Using the E.coli MG1655 genome as a template, the pdhR fragment with adapter was amplified with primers pdhR-F and pdhR-R. Using the ptrc99a plasmid as a template, ptrc99a-ver-F1 and ptrc99a-ver-R1 primers were amplified to obtain the plasmid backbone ptrc99a-Gibson1 for Gibson assembly with adapters, and the above pdhR fragment was obtained by Gibson assembly to obtain plasmid pYR01. The PhdR nucleotide sequence is shown in SEQ NO.2.
[0032] Use the pUC57-GFPmut2 plasmid as a template, with a promoter P AP (SEQ NO.1) The primers GFPmut2-F and GFPmut2-R for the linker amplify the GFPmut2 fragment with the linker. Using the pYR01 plasmid as a template, ptrc99a-ver-F2 and ptrc99a-ver-R2 primers were amplified to obtain a plasmid backbone ptrc99a-Gibson2 for Gibson assembly with a linker, and the above GFPmut2 fragment was used to obtain plasmid pYR02 through Gibson assembly. The plasmid map is as follows ...
Embodiment 2
[0040] Example 2: Testing the pYR02 biosensor
[0041] The plasmid pYR02 was transformed into MG1655 competent medium, and the strain MG1655-pYR02 was constructed. Pick a single colony in 5 mL of LB medium containing ampicillin antibiotics, cultivate overnight at 37°C at 200 rpm to form a seed solution, transfer 100 μL of seed liquid to fresh 5 mL of LB medium containing ampicillin antibiotics, and cultivate at 37°C at 200 rpm until OD ~ 0.2-0.6, different concentrations of PYR were added to achieve the desired different induction concentrations. After induction for 5-7 hours, 200 μL was taken to measure the fluorescence intensity (AFU) and absorbance (OD) at 600 nm of the sample, and the results showed a certain linear relationship between pyruvate and the fluorescence intensity per unit OD.
Embodiment 3
[0042] Example 3: Mutation library construction of PdhR protein
[0043] PdhR protein was mutated using TIANDZ instant error-prone PCR kit v 1.3 to build a library. Specific PCR systems include:
[0044] Table 3 Error-prone PCR reaction system
[0045]
[0046] The error-prone PCR reaction program is: 94°C, 3min; 94°C, 1min, 45°C, 1min, 72°C, 1min, 40 cycles.
[0047]pdhR was connected with the plasmid backbone p15ASI-Gibson3 through Gibson-assemble to form a plasmid mutation library, which was transformed into competent DH5α and coated on a solid plate containing ampicillin resistance. Pick 20 single colonies for sequencing, and the Sanger sequencing of 20 colonies shows that the mutation rate is 0-4 mutations / kb.
[0048] The primers used in this part are as follows:
[0049] Table 4 constructs the primers used in the PYR sensor plasmid mutation library
[0050]
PUM
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