Plasmid carrying green fluorescent protein gene and indicator bacteria with plasmid

Abstract

The invention relates to a plasmid carrying a green fluorescent protein gene and indicator bacteria with the plasmid. The green fluorescent protein gene is expressed in formation, and the plasmid comprises a skeleton sequence, a promoter sequence derived from a salmonella typhimurium rpsM gene and a GFPuv encoding sequence in the downstream of the promoter sequence. Recombination enteritis serotype salmonella contains the plasmid. Recombinant bacteria show a bright green color after ultraviolet excitation, and can be observed with a fluorescence microscope and detected with a flow cytometry and a microplate reader. The recombination bacteria can serve as indicating bacteria of intestinal colonization, organization and cellular localization of salmonella, and can also serve as indicating property control salmonella of the disinfection effect of beast and bird food processing link. The plasmid carrying a green fluorescent protein gene and the indicator bacteria of the plasmid have important functions in aspects of determination of biosafety key points in a food HACCP system.

Description

technical field

[0001] The invention relates to a plasmid carrying a green fluorescent protein gene and an indicator bacterium having the plasmid, in particular to a quality control tracer Salmonella in livestock and poultry slaughtering and processing links and a preparation method thereof, belonging to the technical field of microorganisms. Background technique

[0002] Salmonella is one of the most common causes of human foodborne diseases, and livestock and poultry products carrying Salmonella are an important source of human Salmonella infection and food poisoning. At present, Salmonella of animal origin poses a huge threat to people's health and life safety.

[0003] There are many kinds of Salmonella and they can be classified in various ways. According to the diseases they cause, they can be divided into two categories: Salmonella Typhi that cause typhoid and Nontyphoidal Salmonella that do not cause typhoid. Among the more than 2,500 serotypes that have been discov...

Examples

Embodiment 1

[0043] Example 1 Construction of pCR-rpsM-GFPuv plasmid

[0044] Take the pGFPuv plasmid (purchased from Clontech, see https: / / www.takarabio.com / products / gene-function / fluorescent-proteins / fluorescent-protein-plasmids / cyan-and-green-fluorescent-proteins / gfp for plasmid information -and-gfpuv-fluorescent-proteins website) as template DNA, with PrpsM-gfp-fwd and Pgfp-rev as upstream and downstream primers, and using PrimeSTAR HS DNA polymerase (purchased from TaKaRa Company) to establish a PCR gene amplification reaction, Amplification of the GFPuv coding sequence. At the same time, the PCR reaction also introduces the promoter sequence of the rpsM gene of Salmonella typhimurium at the 5' end of the GFPuv gene by PCR method. Concrete reaction system is as follows:

[0045]

[0046] The PCR reaction conditions were preheated at 95°C for 2 min, followed by 30 cycles of 98°C for 10 s, 55°C for 5 s, and 72°C for 1 min, followed by an extension at 72°C for 5 min. The amplified ...

Embodiment 2

[0050] Example 2 Preparation of Salmonella Competent Cells

[0051] The activated Salmonella SE17 bacteria liquid was inoculated in 100 mL LB liquid medium at a volume ratio of 1:100, and cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.6. Ice bath for 30 minutes to fully cool the bacterial solution; harvest bacterial cells by centrifugation at 4000×g at 4°C for 20 minutes; Resuspend and wash with glycerol; finally resuspend the pellet with 0.2 mL of ice-cold 10% glycerol; aliquot 40 μL / tube to obtain competent cells for Salmonella electric shock transformation.

Embodiment 3

[0052] Example 3 Electric Shock Transformation

[0053] The specific operation steps are:

[0054] (1) Carefully pipette 40 μL of freshly prepared or frozen-thawed Salmonella electric shock transformation competent cells into a 0.5 mL ice-cold micro-sterile centrifuge tube, and pre-cool on ice with the electric shock transformation cup;

[0055] (2) Add 10pg-25ng of plasmid DNA to be transformed (i.e., pCR-rpsM-GFPuv plasmid) in a volume of 1-2μL, and place on ice for 1 min;

[0056] (3) Adjust the electroporator so that the electric pulse is 25μF, the voltage is 25kV, and the resistance is 200Ω; add the mixture of competent cells and plasmid DNA into the ice-cold electric shock cup, put the electric shock cup into the electroporator, and start the electric pulse;

[0057] (4) After the pulse is over, quickly take out the electroshock transformation cup, add 1ml of SOC medium at room temperature, and shake at 37°C at 180 rpm for 2 hours to recover the bacteria;

[0058] (5) ...

technical field

[0001] The invention relates to a plasmid carrying a green fluorescent protein gene and an indicator bacterium having the plasmid, in particular to a quality control tracer Salmonella in livestock and poultry slaughtering and processing links and a preparation method thereof, belonging to the technical field of microorganisms. Background technique

[0002] Salmonella is one of the most common causes of human foodborne diseases, and livestock and poultry products carrying Salmonella are an important source of human Salmonella infection and food poisoning. At present, Salmonella of animal origin poses a huge threat to people's health and life safety.

[0003] There are many kinds of Salmonella and they can be classified in various ways. According to the diseases they cause, they can be divided into two categories: Salmonella Typhi that cause typhoid and Nontyphoidal Salmonella that do not cause typhoid. Among the more than 2,500 serotypes that have been discov...

Examples

Embodiment 1

[0043] Example 1 Construction of pCR-rpsM-GFPuv plasmid

[0044] Take the pGFPuv plasmid (purchased from Clontech, see https: / / www.takarabio.com / products / gene-function / fluorescent-proteins / fluorescent-protein-plasmids / cyan-and-green-fluorescent-proteins / gfp for plasmid information -and-gfpuv-fluorescent-proteins website) as template DNA, with PrpsM-gfp-fwd and Pgfp-rev as upstream and downstream primers, and using PrimeSTAR HS DNA polymerase (purchased from TaKaRa Company) to establish a PCR gene amplification reaction, Amplification of the GFPuv coding sequence. At the same time, the PCR reaction also introduces the promoter sequence of the rpsM gene of Salmonella typhimurium at the 5' end of the GFPuv gene by PCR method. Concrete reaction system is as follows:

[0045]

[0046] The PCR reaction conditions were preheated at 95°C for 2 min, followed by 30 cycles of 98°C for 10 s, 55°C for 5 s, and 72°C for 1 min, followed by an extension at 72°C for 5 min. The amplified ...

Embodiment 2

[0050] Example 2 Preparation of Salmonella Competent Cells

[0051] The activated Salmonella SE17 bacteria liquid was inoculated in 100 mL LB liquid medium at a volume ratio of 1:100, and cultured at 37°C with shaking at 180 rpm until the OD600 reached 0.6. Ice bath for 30 minutes to fully cool the bacterial solution; harvest bacterial cells by centrifugation at 4000×g at 4°C for 20 minutes; Resuspend and wash with glycerol; finally resuspend the pellet with 0.2 mL of ice-cold 10% glycerol; aliquot 40 μL / tube to obtain competent cells for Salmonella electric shock transformation.

Embodiment 3

[0052] Example 3 Electric Shock Transformation

[0053] The specific operation steps are:

[0054] (1) Carefully pipette 40 μL of freshly prepared or frozen-thawed Salmonella electric shock transformation competent cells into a 0.5 mL ice-cold micro-sterile centrifuge tube, and pre-cool on ice with the electric shock transformation cup;

[0055] (2) Add 10pg-25ng of plasmid DNA to be transformed (i.e., pCR-rpsM-GFPuv plasmid) in a volume of 1-2μL, and place on ice for 1 min;

[0056] (3) Adjust the electroporator so that the electric pulse is 25μF, the voltage is 25kV, and the resistance is 200Ω; add the mixture of competent cells and plasmid DNA into the ice-cold electric shock cup, put the electric shock cup into the electroporator, and start the electric pulse;

[0057] (4) After the pulse is over, quickly take out the electroshock transformation cup, add 1ml of SOC medium at room temperature, and shake at 37°C at 180 rpm for 2 hours to recover the bacteria;

[0058] (5) ...

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