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Bicistronic specific DNA utilizing lacZ alpha oligopeptide encoding gene as second gene encoding frame and application of bicistronic specific DNA

A technology of gene coding and DNA molecules, which is applied in the biological field and achieves the effect of great application value

Active Publication Date: 2017-01-25
SHENZHEN PREVENTION & TREATMENT CENT FOR OCCUPATIONAL DISEASES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

n can be specifically 56

Method used

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  • Bicistronic specific DNA utilizing lacZ alpha oligopeptide encoding gene as second gene encoding frame and application of bicistronic specific DNA
  • Bicistronic specific DNA utilizing lacZ alpha oligopeptide encoding gene as second gene encoding frame and application of bicistronic specific DNA
  • Bicistronic specific DNA utilizing lacZ alpha oligopeptide encoding gene as second gene encoding frame and application of bicistronic specific DNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Embodiment 1, construction of recombinant plasmid

[0049] 1. Synthesize the double-stranded DNA molecule shown in sequence 1 of the sequence listing.

[0050] In sequence 1 of the sequence listing, the 9th-14th nucleotides are the recognition sequence of the restriction endonuclease Nde I, the 112th-141st nucleotides are the lac promoter region, and the 146th-168th nucleotides are lac In the control region, the 173rd-177th nucleotides are RBS1, the 186th-188th nucleotides are the start codon of the gene coding frame Ⅰ, and the 189th-206th nucleotides are the MCS region (the sequence is cut by HindⅢ recognition sequence, BamHI restriction recognition sequence and XhoⅠ restriction recognition sequence), the 207th-224th nucleotide is His 6 The coding sequence of the tag, the 225th-227th nucleotide is the stop codon of the gene coding frame I, the 228th-234th nucleotide is RBS2, and the 242-412th nucleotide is the coding sequence of the lacZα short peptide ( The 242nd-24...

Embodiment 2

[0058] Embodiment 2, construct recombinant bacteria

[0059] The recombinant plasmid pBR-ORF-lacZα was introduced into Escherichia coli Top10 to obtain recombinant bacteria I.

[0060] The recombinant plasmid pBR-ORFmu1-lacZα was introduced into Escherichia coli Top10 to obtain recombinant bacteria II.

[0061] The recombinant plasmid pBR-ORFmu2-lacZα was introduced into Escherichia coli Top10 to obtain recombinant bacteria III.

[0062] The recombinant plasmid pBR-ORFmu3-lacZα was introduced into Escherichia coli Top10 to obtain the recombinant strain IV.

[0063] The schematic diagram of the components of each recombinant plasmid is shown in image 3 .

[0064] Rare codons, especially consecutive rare codons, are resistant to gene expression. When rare codons appear in the coding frame of the first gene, the translation level of the coding frame of the second gene will decrease compared to the situation without rare codons, that is, the expression level of the reporter g...

Embodiment 3

[0065] Example 3, Detection of expression of gene coding frame I in recombinant bacteria by color or OD value

[0066] 1. Test treatment

[0067] The recombinant bacteria I, recombinant bacteria II, recombinant bacteria III and recombinant bacteria IV constructed in Example 2 were respectively used as the bacteria to be tested, and the following operations were performed:

[0068] 1. Inoculate the bacteria to be tested into the liquid LB medium containing 50 μg / mL ampicillin, culture at 37°C with shaking at 250 rpm until OD 600nm Value = 0.6.

[0069] 2. After completing step 1, add IPTG and X-gal to the culture system (the concentration of IPTG in the culture system is 0.5mM, and the concentration of X-gal in the culture system is 0.04g / 100ml), shake at 37°C and 250rpm Incubate for 4 hours.

[0070] 3. After completing step 2, take the entire culture system, centrifuge at 3500rpm, take the supernatant, and take pictures.

[0071] 4. Take the supernatant obtained in step 3...

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Abstract

The invention discloses a dicistronic specific DNA utilizing a lacZ alpha oligopeptide encoding gene as a second gene encoding frame and application of the dicistronic specific DNA in expressing a target gene. A specific DNA molecule provided by the invention has a bicistronic structure, and sequentially comprises the following components from upstream to downstream: a lac promoter, a lac control region, a first ribosome bind site, a gene encoding frame I, a second ribosome bind site, a gene encoding frame II and a terminator; the last nucleotide of the gene encoding frame I is connected with a first nucleotide of the second ribosome bind site; and the gene encoding frame II encodes lacZ alph oligopeptide formed by the first to nth amino acid residues from the N terminal of lacZ, wherein n is a natural number within a range of 50-150. The specific DNA molecule provided by the invention has dual capabilities of judging whether an extraneous target gene in the gene encoding frame I is expressed or not and judging the expression amount of extraneous target genes by directly observing without special steps, and has great application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a bicistronic specific DNA with a lacZα short peptide coding gene as a second gene coding frame and its application in expressing a target gene. Background technique [0002] Escherichia coli expression system is the earliest and most widely used exogenous gene expression system in gene expression technology. The incomparable advantages of the system, so E. coli is vividly called "recombinant protein factory". [0003] The expression vector is the core of the E. coli expression system. Generally, a complete expression vector contains multiple necessary elements and can be divided into two categories. The first category is the vector element, which determines the ability of the vector plasmid to exist stably in the host cell. Including: origin of replication (which determines the copy number of the expression vector in the host) and selectable markers (can be used to scr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/56
CPCC12N9/2471C12N15/70C12N2800/101C12N2830/20C12N2840/20C12Y302/01023
Inventor 惠长野郭妍杨学琴郑浩渠高朝贤李丽梅张文黄先青
Owner SHENZHEN PREVENTION & TREATMENT CENT FOR OCCUPATIONAL DISEASES
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