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Electroporation genetic manipulation method of Zymomonas mobilis

A technology of Zymomonas mobilis and Mobilis, which is applied in the field of electroporation genetic manipulation of Zymomonas mobilis, which can solve the problems that are difficult to meet the requirements of Zymomonas mobilis transformation application research, lack of versatility, stability, and repeatability and other issues to achieve the effect of improving conversion efficiency and stability, universality, and reducing restrictions

Inactive Publication Date: 2012-07-25
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] To sum up, the current electroporation transformation methods are all for the special application of specific strains and specific plasmids, and it is difficult to extend to other strains and plasmids. To meet the needs of molecular biology research and further transformation application research, this field needs to establish a more general, efficient and stable electroporation method

Method used

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  • Electroporation genetic manipulation method of Zymomonas mobilis
  • Electroporation genetic manipulation method of Zymomonas mobilis
  • Electroporation genetic manipulation method of Zymomonas mobilis

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1. Electroporation transformation of pBBR1MCS-2 (5144bp) to Zymomonas mobilis strain ZM4 under different cell concentration factors

[0048] The plasmid pBBR1MCS-2 was extracted from Escherichia coli JM110 strain by alkaline method, dissolved in sterile water, and the concentration was adjusted to 150ng / μl to obtain the DNA to be transformed.

[0049] First streak inoculate strain ZM4 onto RM solid medium plate (2% D-glucose, 1% yeast extract, 0.2% KH 2 PO 4 , pH 6.0, agar 1.8%), static culture at 30°C for 3 days, plate activation; pick plate growth single colony, inoculate RM liquid medium (2% D-glucose, 1% yeast extract, 0.2% KH 2 PO 4 , pH 6.0), culture at 30°C for 18 hours, which is a liquid culture; for the above liquid culture, inoculate RM liquid with 1% inoculum, and culture at 30°C until OD 600 =0.405, ice bath for 5min, take 3ml, 6ml, 12ml, 18ml, 24ml respectively into 50ml centrifuge tubes, collect the bacteria by centrifugation at 6000rpm, 4°C, 3m...

Embodiment 2

[0052] Example 2. Electroporation transformation of pZB21 (5930bp) to Zymomonas mobilis strain CP4 under different cell concentration multiples

[0053] The plasmid pZB21 was extracted according to the method in Example 1, and the concentration was adjusted to 750 ng / μl to obtain the DNA to be transformed.

[0054] Activate, cultivate CP4 and prepare competent cells according to the method in Example 1, the difference is OD 600 = 0.40. Add 2 μl of the above-mentioned DNA to be transformed, operate as in Example 1, the electric field strength is 11.75kV / cm (corresponding voltage is 2.35kV), but the recovery time is 12h, and the antibiotic and its concentration are tetracycline Tc1 7.5μg / ml. The results of the corresponding relationship between the obtained concentration multiple and the conversion efficiency are shown in Figure 4 . The transformation efficiency is the highest when the concentration factor is 100 times, reaching 160 / μg DNA.

Embodiment 3

[0055] Example 3. Electroporation transformation of pBBR1MCS-2 (5144bp) into Zymomonas mobilis strain ZM4 under different volumes of competent cells in the electric shock cup

[0056] The plasmid is the same as in Example 1, and the activation and cultivation of ZM4 are the same as in Example 1. OD 600 =0.405 bacteria solution, ice bath for 5min, 6000rpm, 4°C, 3min centrifugation to collect the bacterial cells, remove the supernatant, wash with 20ml of sterile 10% glycerol pre-cooled in ice bath, repeat 3 times, remove the supernatant, and use the above culture 1 / 100 of the volume of the solution, resuspended in ice bath pre-cooled sterile 10% glycerol (that is, the concentration factor is 100 times); according to 40μl, 80μl, 120μl, 160μl, 200μl in each 1.5ml sterile centrifuge tube .

[0057] Add the above-mentioned DNA to be transformed into the above-mentioned dispensing tubes according to the ratio of 1 μl of the above-mentioned DNA to be transformed per 40 μl of cells, ...

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Abstract

The invention discloses an electroporation genetic manipulation method of Zymomonas mobilis, which comprises the following steps: carrying out electroporation to convert DNA (deoxyribonucleic acid) to be converted into competent cells of Zymomonas mobilis, or carrying out incubation on cell-free extract of Zymomonas mobilis and DNA to be converted, and carrying out electroporation to convert the incubated DNA into competent cells of Zymomonas mobilis. In the invention, a cell-free culture liquid of Zymomonas mobilis strains is used for treating the DNA to be converted so as to modify the DNA,thereby enhancing the conversion efficiency; the method disclosed by the invention can be used for carrying out electroporation on different hosts and DNAs by optimizing the key electroporation physical parameters, so as to enhance the conversion efficiency and stability of electroporation genetic manipulation of Zymomonas mobilis and adapt to all the genetic manipulations which are carried out through electroporation conversion and have different construction purposes, so that the method is also universal.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering. Specifically, it relates to an electroporation genetic manipulation method of Zymomonas mobilis. Background technique [0002] The current prominent global energy crisis and environmental pollution have made the industrial production of fuel ethanol using biotechnology and renewable resources (biomass) one of the major research and development projects in many countries, including the United States, Brazil, and China. . These countries are trying to develop new fuel ethanol production strains and processes that can be used for more direct, comprehensive and efficient use of biomass resources. And Zymomonas mobilis ethanol fermentation speed is fast, ethanol yield is high (being 97% of theoretical value, and yeast is 90-92% of theoretical value), can tolerate high sugar concentration and high alcohol concentration, is known at present One of the bacteria with the strongest ethanol-p...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/74
Inventor 邹少兰张鲲洪解放马媛媛井欣张敏华
Owner TIANJIN UNIV