Bacillus subtilislogarithmic phaseexpression system

A Bacillus subtilis, logarithmic phase technology, applied in the field of genetic engineering, can solve the problems of no expression in the stable phase, difficult to achieve early expression, complex determination of the addition phase, etc., to achieve the effect of increasing the concentration of bacteria and increasing the activity of keratinase

Active Publication Date: 2016-12-14
JIANGNAN UNIV
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

At present, researchers mainly use the method of adding inducers to realize the differential expression of the target gene in different periods, and this method has the dis

Method used

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  • Bacillus subtilislogarithmic phaseexpression system

Examples

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Embodiment 1

[0041] Example 1P hag ,P abrB and P valS Acquisition of promoter and construction of expression vector

[0042] Using the Bacillus subtilis 168 genome template and #1 and #2 as primers, the Bacillus subtilis 168 genome P was amplified by PCR hag Promoter DNA fragment; PCR amplification of Bacillus subtilis 168 genome P using Bacillus subtilis 168 genome template, #3 and #4 as primers abrB Promoter DNA fragment; PCR amplification of Bacillus subtilis 168 genome P using Bacillus subtilis 168 genome template, #5 and #6 as primers valS Promoter DNA fragment; reaction conditions: pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s; extension at 68°C for 30s; a total of 34cycles; post-extension for 5min. The above-mentioned three fragments obtained by PCR were treated by double digestion with BamHI and XhoI respectively, and after recovery, they were ligated with pP43 treated with the same digestion by DNA ligase. After transforming into Escherichia coli JM109, the ...

Embodiment 2

[0043] Example 2 Expression of Fluorescent Protein Using Bacillus subtilis Logarithmic Phase Expression System

[0044] Using the plasmid pMD-19T simple-GFP (as shown in SEQ ID NO.15) as a template, and #7 and #8 as primers, clone the gfp fluorescent protein gene (as shown in SEQ ID NO.16), and add XhoI digestion to the front site and RBS, and a PstI restriction site is added to the back end. The PCR products obtained by digestion with XhoI and PstI were ligated to pP43, pPHAG, pPABRB and pPVALS treated with the same enzymes to form new plasmids pP43-gfp, pPHAG-gfp, pPABRB-gfp and pPVALS-gfp.

[0045] (1) Add 10 μL of pP43-gfp, pPHAG-gfp, pPABRB-gfp, and pPVALS-gfp plasmids at a concentration of 100 μg / mL to 500 μL of Bacillus subtilis 168 competent cells, heat shock at 45°C for 3 minutes, and heat at 37°C Shake at 220rpm and incubate for 1 hour, spread on LB plates containing Khanna antibiotics, incubate at 37°C for 10 hours, and verify the grown colonies by colony PCR.

[...

Embodiment 3

[0047] Example 3 Utilize Bacillus subtilis logarithmic phase expression system to express keratinase

[0048] (1) Strain construction: the keratinase gene (shown in SEQ ID NO.17) derived from Bacillus licheniformis (purchased from the American Type Culture Collection, No. ATCC14580) was used as a template with the Bacillus licheniformis genome, #9 and #10 is a primer, and the ker gene fragment is obtained by PCR, with an XhoI restriction site and RBS added to the front end, and a PstI restriction site added to the rear end. The PCR products obtained by digesting with XhoI and PstI were connected to pP43, pPHAG, pPABRB and pPVALS treated with the same enzymes to form new plasmids pP43-ker, pPHAG-ker, pPABRB-ker and pPVALS-ker.

[0049] (2) Enzyme activity comparison: four strains were fermented and cultured, and the enzymatic activity and cell concentration of keratinase were determined by sampling every 2 hours. The results showed that the extracellular enzyme activities of B...

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Abstract

The invention discloses a bacillus subtilis logarithmic phase expression system and belongs to the field of genetic engineering. The expression system includes bacillus subtilis serving as a host and a logarithmic phase started plasmid expression vector. In the culture process of recombinant bacteria, a target gene carried on a plasmid is expressed only after a logarithmic phase begins but before a stable phase. A keratinase gene (ker) is expressed by utilizing the expression system,the thalli concentration and keratinase activityare improved by 10.4-15.2% and 27.8-41.5% respectively through fermentation culture compared with a traditional strongstart-up system.

Description

technical field [0001] The invention relates to a logarithmic phase expression system of Bacillus subtilis, which belongs to the field of genetic engineering. Background technique [0002] Bacillus subtilis (Bacillus subtilis) is playing an important role in applied biology because of its fast growth rate, short fermentation period, and strong ability to secrete extracellular proteins. At the same time, as a well-studied model strain, it is generally recognized as safe (GRAS), and has been widely used in the research and production of food and medicine. [0003] However, when using B. subtilis to overexpress certain metabolites of pathway genes, the expression of precursor synthesis genes promotes the accumulation of precursors to increase yield during the log phase. After entering the stable period, due to energy constraints, the expression of these precursor synthesis genes will cause waste and reduce the yield. In addition, when expressing some enzyme preparations, such...

Claims

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Application Information

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IPC IPC(8): C12N15/75C12N1/21C12R1/125
Inventor 康振陈坚杨森堵国成
Owner JIANGNAN UNIV
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