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Preparation method and transformation method for efficiently transforming competent cells from pichia pastoris

A technology of competent cells and Pichia pastoris, applied in the field of genetic engineering, can solve problems such as failure, achieve the effects of reducing damage, good transformation and expression, and improving permeability

Active Publication Date: 2021-07-23
CUSABIO TECH LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, when the competent state of Pichia pastoris is not good, the transformation will fail when there is a relatively small amount of transformed DNA and a long-length plasmid DNA, or when there is a high content of impurities in the transformed ligation product. How to provide a relatively Optimum competent cells of Pichia pastoris and avoiding transformation failure are the key directions that people in the field pay attention to

Method used

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  • Preparation method and transformation method for efficiently transforming competent cells from pichia pastoris
  • Preparation method and transformation method for efficiently transforming competent cells from pichia pastoris
  • Preparation method and transformation method for efficiently transforming competent cells from pichia pastoris

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Experimental program
Comparison scheme
Effect test

Embodiment approach

[0041] According to a typical embodiment of the present invention, a preparation method of Pichia pastoris competent cells, the method comprises:

[0042] Inoculate Pichia pastoris for the first culture, then transfer to the second culture to obtain the first bacterial liquid;

[0043] performing a third culture on the first bacterial liquid to obtain a second bacterial liquid;

[0044] The second bacterial solution is sequentially resuspended and activated with cell osmotic pressure protection solution, cell membrane permeability enhancement solution, cell stabilization solution and cell DNA protection solution to obtain competent cells.

[0045]Inoculate and cultivate Pichia pastoris, and then transfer culture when the absorbance value OD600 is 1.4-1.7, and obtain the bacterial liquid after transfer culture;

[0046] The culture plate is not easy to exceed two weeks. Pick a large round single colony. It is easier to pick less bacteria. Measure the OD600 value of the seeds b...

Embodiment 1

[0080] Embodiment 1 prepares competent cell

[0081] Take 100ul of bacterial liquid and connect it to a 500ml Erlenmeyer flask with 100ml of YPD medium, 30°C, 250rpm, culture overnight (14-18h), until OD600=0.9-1.4, 1.2 is the best for harvesting, and the harvesting should be rapid . Transfer the bacterial solution to two pre-cooled 50ml centrifuge tubes, each 50ml 3000g, centrifuge at 4°C for 5min, discard the supernatant. Each tube was resuspended with 30ml cell osmotic pressure protection solution, left at room temperature for 30 minutes and then centrifuged, each tube was resuspended with 5ml cell membrane permeability enhancement solution, centrifuged as above, and the supernatant was discarded. Resuspend each tube with 5ml ice-sterile water, centrifuge as above, and discard the supernatant. Each tube was resuspended with 5ml ice 1.2M sorbitol, centrifuged as above, and the supernatant was discarded. Each tube was resuspended with 500ul cell DNA protection solution, an...

Embodiment 2

[0085] Embodiment 2 electrotransformation and PCR amplification

[0086] Preheat the Eppendorf electroporator, adjust the parameters to 1.5kV, 5mS; take a freshly prepared or 65ul electrocompetent cells frozen at -80°C within two weeks, add 20ul linearized pPIC9k (PPIC3.5K) recombinant plasmid (about 10ug), such as figure 1 , mixed gently, transferred to a pre-cooled 0.2cm Eppendorf electro-cup, gently tapped on the ultra-clean bench to make the mixture flow to the bottom of the electro-shock cup, and ice-bathed for 5 minutes. (Do not leave the ice for all operations); immediately add a total volume of 1mL pre-cooled 1molL sorbitol to the electric transfer cup within 5S, and transfer the contents to sterilized 1.5mL centrifuge tubes; gently take out with a 1ml pipette tip Spread 200ul on the RDB plate; culture it upside down at 30°C for 3-5 days, and observe the appearance of transformants on the plate.

[0087] PCR identification: Randomly mark 1-20 single spots on the prep...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a preparation method and a transformation method for efficiently transforming competent cells from pichia pastoris, and the method comprises the following steps: inoculating pichia pastoris for a first culture, and then transferring for a second culture to obtain a first bacterial liquid; performing a third culture on the first bacterial liquid to obtain a second bacterial liquid; the second bacterial liquid is subjected to resuspension activation with a cell osmotic pressure protection liquid, a cell membrane permeability enhancement liquid, a cell stabilizing liquid and a cell DNA protection liquid in sequence, and competent cells are obtained. According to the competent celsl obtained by the method, the activity of the competent cells is 85%-95%, the competent cells have extremely high transformation efficiency, and 2*10 <3> transformants can be obtained by transforming the competent cells with plasmids (the concentration is 5[mu]g / [mu]l); the success rate of PCR identification reaches 90% or above.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a preparation method and a transformation method for efficiently transforming competent cells of Pichia pastoris. Background technique [0002] The methanol yeast expression system has many advantages, among which the Pichia yeast expression system is the most well-known and widely used in the expression of foreign proteins. The expression operation of Pichia pastoris is simple and the expression level is high, but in actual operation, not every exogenous gene can be successfully expressed at a high level. [0003] Pichia pastoris is a yeast species that can efficiently express recombinant proteins. On the one hand, because it belongs to eukaryotes, the expressed protein can be modified by glycosylation. On the other hand, Pichia pastoris The growth rate is fast, and the expressed protein can be secreted into the medium, which is convenient for protein pu...

Claims

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Application Information

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IPC IPC(8): C12N1/16C12N15/81C12R1/84
CPCC12N1/16C12N1/005C12N15/815
Inventor 廖怡辉许静兰陈思龙符磊
Owner CUSABIO TECH LLC
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