Method for improving acid stress resistance of recombinant escherichia coli
A technology for recombining Escherichia coli and Escherichia coli, which is applied in the field of microbial engineering to achieve the effect of improving acid stress resistance and simple operation
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Embodiment 1
[0036] Example 1: Construction of recombinant strain E.coli K12 MG1655 / pTrc99a-TreC
[0037] Specific steps are as follows:
[0038](1) Based on the treC gene sequence in the NCBI database (the TreC gene encoding trehalose 6-phosphate hydrolase, involved in the metabolic pathway of trehalose 6-phosphate, the process of regulating the hydrolysis of 6-phosphate in the trehalose system) is designed as SEQ Primers pTrc99a / TreC-F and pTrc99a / TreC-R shown in ID NO.2 and SEQ ID NO.3;
[0039] (2) designing the primer loop p-pTrc99a-F and the loop p-pTrc99a-R shown in SEQ ID NO.4 and SEQ ID NO.5 respectively;
[0040] (3) Using the genome of E.coli K12 MG1655 as a template, using p-pTrc99a / TreC-F, p-pTrc99a / TreC-R as primers to obtain the gene fragment shown in SEQ ID NO.1 by PCR amplification, and obtain the PCR product;
[0041] (4) Using the vector pTrc99a as a template, using loop p-pTrc99a-F and loop p-pTrc99a-R as primers to obtain a linearized long fragment of the vector by ...
Embodiment 2
[0045] Embodiment 2: the growth situation of recombinant bacterial strain and control bacterial strain under normal conditions
[0046] Specific steps are as follows:
[0047] (1) The bacterial strains E.coli K12 MG1655 / pTrc99a-TreC, E.coliK12MG1655 / pTrc99a-TreB obtained in Example 1 and the control bacterial strain E.coli K12 MG1655 / pTrc99a were respectively inoculated in LB liquid medium for activation, and placed Cultivate at 220rpm in a shaker at 37°C for 12h to obtain seed liquid;
[0048] (2) Transfer the seed solution obtained in the above step (1) to LB liquid medium with an inoculation amount of 2% (v / v), and place it in a shaker at 37°C at 220rpm for cultivation; sampling every 2 hours , measure the OD value under the 600nm wavelength, and draw the growth curve (the growth curve obtained by drawing is as follows: figure 1 ).
[0049] The result is as figure 1 As shown, through the analysis of the growth performance test, after 12 hours of cultivation, the growth ...
Embodiment 3
[0051] Example 3: Tolerance test of recombinant strain E.coli K12 MG1655 / pTrc99a-TreC under itaconic acid stress (pH 4.2)
[0052] Specific steps are as follows:
[0053] (1) The control strain E.coli K12 MG1655 / pTrc99a and the recombinant strain E.coliK12MG1655 / pTrc99a-TreC obtained in Example 1 were respectively inoculated in LB liquid medium for activation, and cultured in a shaker at 37°C at 220rpm for 12h , to obtain the seed solution;
[0054] (2) Transfer the seed solution obtained in the above (1) to fresh LB liquid medium with an inoculum amount of 2% (v / v), culture at 220rpm in a shaker at 37°C for 4.5h, and cultivate to logarithm In the mid-growth period, the OD600 at this time is 1.4-1.5, and the culture medium is obtained;
[0055] (3) Centrifuge the culture solution obtained in step (2) for 5 min at 6000 rpm, collect the thalline, wash the thalline obtained twice with 0.85% PBS buffer solution, and resuspend in an equal volume of fresh clothing In conic acid L...
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