Yunnan red pear-1 metallothionein gene PyMT1 and prokaryotic expression vector and application thereof
A metallothionein, prokaryotic expression technology, applied in metallothionein, application, genetic engineering and other directions, to achieve the effect of improving the ability to tolerate heavy metals and improving the tolerance of microorganisms to heavy metal ions
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Embodiment 1
[0038] Example 1: Prokaryotic expression vector pGEX-4t-1- PyMT1 And pET-32a- PyMT1 Build
[0039] Prokaryotic expression vector pGEX-4t-1- PyMT1 、 p ET-32a- PyMT1 The construction process is as figure 1 with figure 2 As shown, first, according to the full-length gene sequence of the exocarp EST of Yunhong Pear No. 1 in GenBank, a pair of primers was designed, and the first strand cDNA of Yunhong Pear No. 1 was used as a template to amplify. PyMT1 Full-length cDNA sequence, recovered and purified PyMT1 After the full-length gene fragment, connect it to the pMD18-T vector to obtain pMD18- PyMT1 ;use EcoR I and Xho I double digestion pMD18- PyMT1 , PGEX-4t-1 and pET32a- xyk , Recovery and purification PyMT1 Gene fragments and large vector fragments pET-32a and pGEX-4t-1, then ligate to obtain recombinant plasmid pGEX-4t-1- PyMT1 And pET32a-PyMT1, which contains the promoter lac, followed by PyMT1 gene. The specific construction process is as follows:
[0040] 1. Yunhongli No. 1 ...
Embodiment 2
[0063] Example 2: Prokaryotic expression vector pGEX-4t-1- PyMT1 , PET-32a- PyMT1 Prokaryotic expression of
[0064] Take 500μl of the fusion expression engineering bacteria solution and add it to 50ml LB liquid medium, and culture for 2 hours at 37℃, 180 rpm shaker; after the cultivation, take out 1 ml, measure the OD600 value of the bacterial solution, and take it out. 2 tubes (2ml / tube) of bacterial solution, as an uninduced sample, add 0.1M IPTG (final concentration of 1 mM and 0.5 mM) to the remaining bacterial solution in the Erlenmeyer flask for induction, and culture in a shaker at 37°C and 180 rpm At 2h, take out 2 tubes (2ml / tube) of bacterial solution, which are samples for 2h induction; also take 2 tubes of samples at induction 4h, 6h, 8h.
[0065] After sampling, one tube of samples for 0, 2, 4, 6, and 8 hours of induction was taken and used for total protein analysis. The specific steps are: centrifugation at 4°C, 12000 rpm for 2 min; discard the supernatant, add 10...
Embodiment 3
[0075] Example 3: Fusion expression engineering strain pGEX-4t-1- PyMT1 BL21 and control bacteria pGEX-4t-1 BL21 heavy metal tolerance experiment
[0076] The experiment was performed on the fusion expression engineering strain pGEX-4t-1- under 37℃, IPTG 0.25 mM / L induction conditions PyMT1 BL21 and the control strain pGEX-4t-1 BL21 carried out heavy metal tolerance experiments. The experimental results showed that the fusion expression engineering strain pGEX-4t-1- PyMT1 BL21 at 1.1 mM / L Zn 2+ When processing, PyMT11 There is no difference between the fusion expression engineering bacteria and the negative control bacteria of the empty vector at 0-5 h, but after 5 h PyMT11 Accelerated growth of transformants ( Picture 12 ). Using 2.25 mM CuCl 2 After treatment, within 0-4 h, PyMT11 Both the transformed bacteria and the negative control grow, within 5-9 h PyMT11 The transformed bacteria still grew slowly, while the negative control showed growth arrest ( Figure 13 ). ...
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