High-temperature cutinase and gene order thereof
A technology of cutinase and high temperature, applied in the direction of genetic engineering, plant gene improvement, hydrolytic enzymes, etc., can solve the problems of obtaining a large number of products, the inability to use gene recombination technology, and insufficient attention to the research of bacterial cutinase, achieving high stability, The effect of efficient expression
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0041] This example illustrates the extraction of total DNA from Thermobifida fusca WSH03-11.
[0042] The Thermobifida fusca WSH03-11 strain was cultured in LB liquid medium (peptone 10g / L, yeast extract 5g / L, NaCl 10g / L) for 2 days, collected by centrifugation at 10000rpm, washed with sterile water, and the collected precipitate was suspended in 500μL Tris -EDTA (trishydroxymethylaminomethane-ethylenediaminetetraacetic acid) buffer solution, add 15 μL lysozyme, incubate at 37°C for 30 minutes, then add 5 μL RNase, incubate at 37°C for 30 minutes, add 30 μL 10% SDS (dodecane Sodium sulfate) and 15 μL proteinase K, incubated at 37°C for 60 min, added 100 μL NaCl (5M) and 80 μL CTAB (cetyltrimethylammonium bromide), incubated at 65°C for 20 min, and incubated with 700 μL of phenol:chloroform: Extract with a mixed solvent of isoamyl alcohol at a volume ratio of 25:24:1, centrifuge at 10,000 rpm, extract the supernatant with 700 μL of chloroform:isoamyl alcohol at a volume ratio ...
Embodiment 2
[0044] This example illustrates the cloning procedure for the gene encoding a high-temperature cutinase.
[0045] Thermobifida fusca WSH03-11 total DNA was used as a template, the following nucleotide sequences were used as primers, and the underlined restriction sites Nco I and EcoR I were used to amplify the high-temperature cutinase gene by PCR.
[0046] Primer 1: 5'-ggAATACCATATgT CCATgg CCAACCCCTACgAgCgCgg-3'
[0047] Primer 2: 5'CATCTCgAgA wxya gggAACgggCAggTggAgCg-3'
[0048] The PCR reaction was carried out in a 50 μL system. The reaction conditions were 35 cycles of denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 1 minute. A 780bp PCR fragment was amplified and recovered by tapping the rubber. The recovered fragment was ligated with the pMD18-Tsimple vector, the ligated product was transformed into Escherichia coli JM109, and the transformed product was coated on an LB plate containi...
Embodiment 3
[0050] This example illustrates the modification procedure of the high temperature cutinase gene.
[0051] Since there is an Nco I restriction site inside the target gene, and the two ends of the gene are respectively Nco I and EcoR I sites, primers were designed to remove the mutation of the Nco I site to facilitate the cloning and expression in the next step. Use the CUT-pMD18-T simple linked to the cutinase gene as a template for site-directed mutagenesis PCR, and design primers:
[0052] Primer 3: 5'-CATgggCCACTCAATgggCggCggCggC-3'
[0053] Primer 4: 5'-gCCgCCgCCgCCCATTgAgTggCCCATg-3'
[0054] The PCR reaction was carried out in a 50 μL system. The reaction conditions were 35 cycles of denaturation at 95°C for 5 minutes, denaturation at 95°C for 1 minute, annealing at 55°C for 1 minute, and extension at 72°C for 4 minutes. The PCR product was transformed into E.coli JM109 competent cells. The transformed bacteria were coated with 100mg / L ampicillin LB plate, and a singl...
PUM
| Property | Measurement | Unit |
|---|---|---|
| molecular weight | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to view more - R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap