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Method for improving electric transformation efficiency of genus cupriavidus bacteria with high GC content

A technology of copper-eating bacteria and electrotransformation, applied in the field of genetic engineering, can solve the problems of low transformation efficiency, inability to transform, and low transformation efficiency, and achieve the effect of improving electrotransformation efficiency

Active Publication Date: 2019-02-22
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conventional bacterial transformation methods include chemical transformation and electrotransformation. Electrotransformation is widely used for its simplicity and high efficiency, but these methods are generally only applicable to model organisms such as E. For bacteria with high GC content, surface-abundant extracellular polymeric substances (EPS) make transformation less efficient or even impossible

Method used

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  • Method for improving electric transformation efficiency of genus cupriavidus bacteria with high GC content
  • Method for improving electric transformation efficiency of genus cupriavidus bacteria with high GC content
  • Method for improving electric transformation efficiency of genus cupriavidus bacteria with high GC content

Examples

Experimental program
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Effect test

Embodiment 1

[0038] Example 1 Plasmid pTR102:: LuxAB electroporation into Cupriavidus taiwanensis X1

[0039](1) Cultivation of the recipient bacteria: Take a ring of Cupriavidus taiwanensis X1 from the cryopreservation tube and culture it on LB solid medium at 37°C for 24 hours at a constant temperature, pick the activated single colony, and inoculate it in 100ml containing 1% v / v spit Warm 80 and 100U / mL pectinase in 20% LB liquid medium, shake culture at 37°C until OD 600 = about 0.6, then centrifuge at 6000 g at 4°C for 15 min to collect the bacteria, resuspend and wash twice with sterile deionized water.

[0040] (2) Preparation of competent cells: resuspend the cells obtained in step (1) in 10% pre-cooled glycerol, centrifuge at 5000g at a constant temperature for 10 minutes at 4°C, remove the supernatant to collect cells, repeat this operation once, and obtain The bacteria were resuspended in 1ml of 10% pre-cooled glycerol, and the obtained competent cells could be directly used fo...

Embodiment 2

[0049] Example 2 Plasmid pTR102::GFP electroporation into Cupriavidus taiwanensis X1

[0050] The difference from Example 1 is that the plasmid pTR102::LuxAB in step (3)b was replaced with pTR102::GFP.

[0051] The construction method of plasmid pTR102::GFP is as follows: 1) using the commercialized plasmid PPK2-OSCA-GFP as a template to amplify the GFP gene; 2) using restriction enzymes PstI and KpnI to double-digest pTR102::LuxAB; 3) The amplified GFP gene was inserted into the large fragment recovered from pTR102::LuxAB after double enzyme digestion, and the plasmid pTR102::GFP was constructed.

[0052] Plasmid pTR102::GFP contains the green fluorescent protein gene GFP. Bacteria successfully transformed into the plasmid can not only grow on corresponding antibiotic-resistant plates, but also emit green fluorescence under a fluorescent microscope, as shown in figure 2 shown.

[0053] In this embodiment, the electrical conversion efficiency can reach 2.0×10 4 cfu / ug~2.4×...

Embodiment 3

[0054] Example 3 Plasmid pTR102:: GFP electroporation into Cupriavidus gilardii T-1

[0055] The difference from Example 2 is that the Cupriavidus taiwanensis X1 in step (1) is replaced by Cupriavidus gilardii T-1.

[0056] Plasmid pTR102::GFP contains the green fluorescent protein gene GFP. Bacteria successfully transformed into the plasmid can not only grow on corresponding antibiotic-resistant plates, but also emit green fluorescence under a fluorescent microscope, as shown in image 3 shown.

[0057] In this embodiment, the electrical conversion efficiency can reach 1.7×10 4 cfu / ug~1.9×10 4 cfu / ug.

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Abstract

The invention provides a method for improving the electric transformation efficiency of genus cupriavidus bacteria with high GC content. The method comprises the following steps: after activating thegenus cupriavidus bacteria with high GC content, inoculating into a diluted eutrophic culture medium containing a surfactant and pectinase; after culturing for a period of time, centrifuging and collecting thalli; then re-suspending and washing with sterile de-ionized water for a plurality of times; centrifuging and collecting thallus sediment; re-suspending in a pre-cooled glycerol water solutionto prepare competent cells and carrying out electric transformation. According to the method provided by the invention, the culture medium is improved to find out that extracellular polymers of the genus cupriavidus bacteria can be effectively reduced by reducing the content of nutrient substances in the culture medium and adding a proper amount of the surfactant and pectinase, so that the electric transformation efficiency of the competent cells is greatly improved. The invention develops a stable and efficient electric transformation method aiming at the characteristics of the genus cupriavidus bacteria with high GC content, and provides powerful technical supports for deeply researching and analyzing the genus cupriavidus bacteria.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for improving the electrotransformation efficiency of copper-phager bacteria with high GC content. Background technique [0002] The genus Cupriavidus is a genus in the taxonomy of bacteria, which currently includes 17 species, namely: C.alkaliphilus, C.basilensis, C.campinensis, C.gilardii, C.laharis, C. C. metallidurans, C. necator, C. nantongensis, C. numazuensis, C. oxalaticus, C. pampae, C. pauculus, C. pinatubonensis, C. plantarum, C. respiraculi, C. taiwanensis, C. yeoncheonensis. The bacteria it belongs to are Gram-negative bacteria with rod-shaped, obligate aerobic, motile, flagella and other characteristics, and are resistant to copper, cobalt, zinc and other metals. The bacteria belonging to this group were mostly isolated from soil, water, pond sediments, legume nodules and lahar sediments. According to the analysis of the genome seq...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74
CPCC12N15/74
Inventor 方连城秦晗王尧吴祥为花日茂
Owner ANHUI AGRICULTURAL UNIVERSITY
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