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Gene induced expression system related to bacteriophage

A technology of gene induction and expression system, which is applied in the field of biology, phage infection and gene induction expression, to achieve the effect of specific induction of expression intensity and reduction of background

Pending Publication Date: 2022-05-27
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Since it is difficult to determine which part of the bacteria in the culture system is infected and when it is infected, it is difficult to achieve such induction purpose by artificially adding small molecule inducers and other non-specific methods.

Method used

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  • Gene induced expression system related to bacteriophage
  • Gene induced expression system related to bacteriophage
  • Gene induced expression system related to bacteriophage

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0081] Example 1 Preparation of phage IP1_SPT7

[0082] 1) The host bacteria host8 obtained by transforming CCP1-174T7 into FM15 bacteria was prepared into competent cells for subsequent use.

[0083] Prepare a 10cm non-resistant LB solid culture plate for later use.

[0084] Prepare LB semi-solid medium containing 0.6% (w / v) agar, 15 μg / mL tetracycline and 50 μg / mL carbenicillin, and preheat at 50°C for use.

[0085] 2) The host bacterium host8 single clone was cultured overnight in LB liquid medium containing 15 μg / mL tetracycline and 50 μg / mL carbenicillin. The culture conditions were 37°C, 200 rpm.

[0086] 3) The overnight cultured host bacteria host8 was diluted 100 times in the same medium preheated at 37°C, and the same conditions continued to activate and cultivate to log phase OD.600 ≈0.3. (Use 1cm cuvette to detect)

[0087] 4) Continue to dilute 20-fold with the same medium preheated at 37°C for the log-phase host bacteria host8, and continue to activate and cu...

Embodiment 2

[0092] Example 2 Phage IP1_SPT7 amplification.

[0093] 1) The host bacterium host8 monoclonal was cultured overnight in M9 liquid medium containing 1% (w / v) CAA, 0.4% (w / v) glucose, 15 μg / mL tetracycline, 50 μg / mL carbenicillin, and the culture conditions 37°C, 200rpm.

[0094] 2) The overnight cultured host bacteria host8 was diluted 100 times in the same medium preheated at 37°C, and the same conditions continued to activate and cultivate to log phase OD. 600 ≈0.3. (Use 1cm cuvette to detect)

[0095] 3) Continue to dilute 20-fold with the same medium preheated at 37°C for the log-phase host bacteria host8, and continue to activate and cultivate to the log-phase OD under the same conditions. 600 ≈0.3. (Use 1cm cuvette to detect)

[0096] 4) Dilute the activated host8 100-fold in the same medium to a volume of 2 mL. Then, the plaques formed by the phage IP1_SPT7 in "Example 1" were inoculated into the diluted host8 host bacteria with an inoculation loop, and then cultu...

Embodiment 3

[0098] Example 3 Phage IP1_SPT7 titer determination

[0099] 1) Prepare 8 10cm non-resistant LB solid culture plates for use (the number of plates can be increased or decreased according to the subsequent phage dilution gradient)

[0100] Prepare LB semi-solid medium containing 0.6% (w / v) agar, 15 μg / mL tetracycline and 50 μg / mL carbenicillin, and preheat at 50°C for use.

[0101] 2) Use the same method in "Example 1" to activate the host bacteria host8 for two rounds to log phase for subsequent use.

[0102] 3) Dilute the phage stock solution obtained in "Example 2" by 10 4～11 fold a total of 8 gradients. (The specific dilution gradient can be increased or decreased according to actual needs, but each gradient requires a 10cm non-resistant LB solid culture plate)

[0103] 4) Mix 10 μL of each of the diluted IP1_SPT7 phages with 200 μL of log-phase host8 host bacteria, add 4 mL of the prepared LB semi-solid medium, and coat the samples on different solid culture plates afte...

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Abstract

The invention relates to a phage-related gene induced expression system, and particularly discloses a gene induced expression system which comprises host bacteria and a phage, wherein the host bacteria comprise a target gene, and the bacteriophage comprises a regulatory gene; the regulatory gene is a gene capable of expressing a regulatory factor after bacteriophage infects host bacteria, and the regulatory factor obtained by expression of the regulatory gene can specifically regulate a promoter of a target gene, or can specifically regulate expression of the target gene or modification after expression. According to the expression system, bacteriophage infection serves as an induction factor for the first time, expression of downstream target genes is started, and then expression of target genes or signal genes in specific host bacteria is achieved. The method provided by the invention not only realizes the expression of the target protein or the signal protein, but also realizes the expression of the target protein or the signal protein in the specific host bacteria, so that the host bacteria in the same culture system are distinguished based on whether the host bacteria are infected by the bacteriophage or not.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to the field of phage infection and gene induction expression. Background technique [0002] Inducible gene expression is a common gene control technology in the field of biotechnology. By adding small molecule inducers to bacteria or cell culture systems, the repressed state of the gene of interest is released, and its expression is activated. For example, in lacoperon, the inhibitory effect of the repressor protein lacI on the lac promoter is released by adding an IPTG inducer, thereby initiating the expression of the lacZ gene. For another example, in the gene circuit controlled by riboswitches, mRNA suppresses its own translation and expression by folding into a special secondary structure. The corresponding ligand can bind to mRNA to unravel its secondary structure and initiate translation. So adding small molecule ligands can induce riboswitch-controlled gene expression...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/73C12N15/74C12N15/63G01N21/64C12R1/19C12R1/01
CPCC12N15/73C12N15/74C12N15/635G01N21/6486G01N21/64C12N15/63C12R2001/01C12R2001/19C12P21/02C12N15/70
Inventor 刘陈立赖旺生魏婷陈茜孙陈健
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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