Preparation method for synthesizing biosensor by using lycopene operon as well as corresponding biosensor and application thereof

A technology of lycopene and synthetic biology, which is applied in the fields of genetic engineering and molecular biology, to achieve the effects of high safety, convenient use, and improved synthesis efficiency

Active Publication Date: 2021-06-01
QINGDAO AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report on the use of lycopene for visual detection of explosive molecules

Method used

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  • Preparation method for synthesizing biosensor by using lycopene operon as well as corresponding biosensor and application thereof
  • Preparation method for synthesizing biosensor by using lycopene operon as well as corresponding biosensor and application thereof
  • Preparation method for synthesizing biosensor by using lycopene operon as well as corresponding biosensor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1: Gene acquisition and vector construction

[0040] 1. Gene acquisition

[0041]The lycopene operon (Addgene NO.53270) from Erwinia agglomerans, whose nucleotide sequence is shown in SEQ ID NO.1, was chemically synthesized on the pUC-57 vector by Golden Wisdom Company to obtain pUC-lyc vector.

[0042] The promoter yqjF (GenBank accession number is AAC76136.1) from Escherichia coli, whose nucleotide sequence is shown in SEQ ID NO.2, was chemically synthesized by Jinweizhi Company on the pUC-57 carrier to obtain pUC-yqjF vector.

[0043] 2. Construction of pET-lyc expression vector

[0044] Using pUC-lyc as a template, primer crtI-pET-F and primer crtE-pET-R, carry out polymerase chain reaction (PCR), amplify lyc fragment, and its PCR amplification system is as follows:

[0045]

[0046] The PCR program was: 95°C 3min; 30 cycles × (95°C 15s, 58°C 15s, 72°C 3min); 72°C 5min; 16°C ∞.

[0047] The primer sequences are as follows:

[0048] crtI-pET-F:

[0...

Embodiment 2

[0080] Example 2: Construction of XLYC1 recombinant strain

[0081] The pET-yqjF-lyc plasmid, pACYC-MvaE-MvaS-GPPS plasmid (cited from Yang J, Nie Q, Ren M, et al. Metabolic engineering of Escherichia coli for the biosynthesis of alpha-pinene. Biotechnology For Biofuels. 2013, 6 : 60.) and pTrc-low plasmid (cited from YangJ, Xian M, Su S, et a1. Enhancing production of bio-isoprene using hybrid MVApathway and isoprene synthase in E.coli.PLoS One.2012; 7:e33509.) Co-transform E.coli BL21(DE3) competent cells, spread on LB solid plates containing 34mg / L chloramphenicol, 100mg / L ampicillin and 30mg / L kanamycin to obtain positive clones, thus obtaining The engineered strain XLYC1 containing the vectors pET-yqjF-lyc, pACYC-MvaE-MvaS-GPPS plasmid and ptrc-low plasmid.

Embodiment 3

[0082] Example 3: Application of Visual Detection of Explosive Molecules

[0083] Conical flask detection: Pick the single colony of engineering Escherichia coli XLYC1 obtained in Example 2 into 10 mL of LB liquid medium containing 34 mg / L chloramphenicol, 100 mg / L ampicillin and 30 mg / L kanamycin, and place it at 37 ℃ Shaker overnight for activation; then transferred to 100mL M9 medium for expansion, and cultured to OD 600 For 0.6-0.8, add 0.5mM isopropyl-β-D-thiogalactoside (IPTG) for induction, add 50mg / L DNT to the positive control, incubate at 30°C on a shaker, and take pictures.

[0084] Anaerobic tube detection: Pick the single colony of engineering Escherichia coli XLYC1 obtained in Example 2 into 10 mL of LB liquid medium containing 34 mg / L chloramphenicol, 100 mg / L ampicillin and 30 mg / L kanamycin, and put it in 37 mg / L LB liquid medium. Incubate at ℃ in a shaker overnight for activation, then transfer to 2 mL of M9 medium, and cultivate to OD. 600 For 0.6-0.8, add...

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Abstract

The invention discloses a preparation method for synthesizing a biosensor by using a lycopene operon as well as the corresponding biosensor and application thereof. The biosensor comprises a recombinant plasmid containing a lycopene operon, a mevalonic acid pathway upstream expression vector and a mevalonic acid pathway downstream expression vector; the recombinant plasmid contains a lycopene operon and a promoter, wherein the nucleotide sequence of the lycopene operon is as shown in SEQ ID NO.1 or the lycopene operon is as shown in SEQ ID NO.1, has more than 90% of homology with the nucleotide sequence as shown in SEQ ID NO.1 and can synthesize the nucleotide sequence of lycopene, and the promoter is as shown in SEQ ID NO.2. The biosensor can sense explosive molecules with different concentrations to synthesize lycopene with different yields, the concentration of the explosive molecules and the yield of the lycopene are coupled, visual detection of the explosive molecules is achieved, the detection operation is simple and convenient, and the safety is high.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering and molecular biology, and in particular relates to a preparation method for synthesizing a biosensor by utilizing lycopene operon, and a corresponding biosensor and application thereof. Background technique [0002] Biosensing technology uses genetic engineering to transform strains, so that after the microorganism senses specific compounds or metabolites of specific compounds in microorganisms, the microorganisms undergo changes that can be detected, so as to achieve the purpose of detecting specific compounds. This biosensor is mainly composed of two parts: a sensing element and a reporting element. The sensing element can specifically sense the target compound. The sensing element is the promoter, ribosome binding site, terminator, transcriptional regulator, etc. responsible for gene transcription. The reporter element can generate a sensing signal under the action of the sensing e...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N15/66C12N1/21G01N27/447C12R1/19
CPCC07K14/195C12N9/0006C12N9/1025C12N9/1029C12N9/1205C12N9/1229C12N9/88C12N9/90C12N15/66C12N15/70C12Y101/01088C12Y203/01009C12Y203/0301C12Y207/01036C12Y207/04002C12Y401/01033C12Y503/03002G01N27/44747
Inventor 杨建明李美洁吕书喆王兆宝
Owner QINGDAO AGRI UNIV
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