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Method for Improving Electrotransformation Efficiency of High GC Content Copperphagia Bacteria

A copper-phagocytic bacteria and electrotransformation technology, applied in the field of genetic engineering, can solve the problems of inability to transform, low transformation efficiency, low transformation efficiency, etc., and achieve improved electrotransformation efficiency, reduced mutual adhesion, and reduced production of extracellular polymers Effect

Active Publication Date: 2022-02-22
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 Electrotransformation Efficiency of High GC Content Copperphagia Bacteria
  • Method for Improving Electrotransformation Efficiency of High GC Content Copperphagia Bacteria
  • Method for Improving Electrotransformation Efficiency of High GC Content Copperphagia Bacteria

Examples

Experimental program
Comparison scheme
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 electrotransformation efficiency of bacteria of the genus Cuferophage with high GC content. After activation, the bacteria of the genus Cuferophage with high GC content are inoculated in a diluted nutrient-rich medium containing a surfactant and pectinase, After culturing for a period of time, the cells were collected by centrifugation, then resuspended and washed with sterile deionized water several times, the cell pellet was collected by centrifugation, resuspended in pre-cooled glycerol aqueous solution, and the prepared competent cells were electroporated. In the present invention, by improving the culture medium, it is found that reducing the content of nutrients in the culture medium and adding an appropriate amount of surfactant and pectinase can effectively reduce the production of extracellular polymers of bacteria of the genus Copperphaga, greatly improving the production capacity of competent cells. Electric conversion efficiency. The invention develops a stable and high-efficiency electrotransformation method aiming at the characteristics of copper-phager bacteria with high GC content, and provides powerful technical support for in-depth research and analysis of copper-phager 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 Patents(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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