Application of sll0147 gene in synthesizing synechocystis carotenoids
A technology of carotene and Synechocystis, applied in the application field of zeaxanthin production, to achieve the effect of enhancing biomass and increasing carotenoid content
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0049] The isolated and cloned sll0147 gene cDNA fragment, the psbA2 promoter fragment, the upstream fragment of homologous recombination slr1285UcDNA fragment and the upstream fragment of homologous recombination slr1285DcDNA fragment were used to construct the overexpression vector of Synechocystis sp. PCC6803sll0147 gene.
[0050] Cloning of Synechocystis sp. PCC6803 psbA2 promoter fragment: according to GenBank landing Synechocystis sp. PCC6803 (accession number: BA000022, AP012205), the first 500 bp of psbA2 ORF was used as the promoter sequence, and primers were designed:
[0051] Promoter-SalI-F: 5'-AATGTCGACTGCCCAGATGCAGGCCTTC-3';
[0052] Promotor-R: 5'-GTAGAGCAGTTCACGCATTTGGTTATAATTCCTTAT-3';
[0053] The amplification procedure is as follows:
[0054] Pre-denaturation at 94°C for 5min, denaturation at 94°C for 30s, renaturation at 55°C for 30s, extension at 72°C for 1min, after 35 cycles, 10min at 72°C, and storage at 4°C.
[0055] After the PCR reaction, the gel ...
Embodiment 2
[0081] Construction of Synechocystis sp. PCC6803sll0147 gene overexpression vector:
[0082] Preparation of plasmid pBluscriptSKplus-T1T2:
[0083] The plasmid pKK233-2 (purchased from Clontech Company) that amplifies the Escherichia coli T1T2 terminator, introduced a PstI restriction site at its 5' end, and introduced a BamHI restriction site at its 3' end, and the primers used were:
[0084] T1T2-F: 5'-ATACTGCAGCCAAGCTTGGCTGTTTTGGC-3';
[0085] T1T2-R: 5'-TTAGGATCCCCCATTATTGAAGCATTTAT-3';
[0086] The amplification procedure is as follows:
[0087] Pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30s, renaturation at 60°C for 30s, extension at 72°C for 30s, after 35 cycles; extension at 72°C for 10 minutes, and storage at 4°C; the obtained gene fragments were digested with PstI and BamHI and the same The digested pBluescriptSK was ligated to obtain plasmid pBluescriptSKT1T2.
[0088] The homologous recombination upstream arm slr1285U gene fragment obtaine...
Embodiment 3
[0104] PCR detection of sll0147 gene overexpressed algal strain
[0105] Using transgenic Synechocystis PCC6803 and wild-type Synechocystis PCC6803 containing the expression vector p5S1285UDs110147 as materials, the total DNA was extracted for PCR detection and analysis. The specific method is as follows:
[0106] Neutral phenol reagent (purchased from Invitrogen) was used to extract DNA from sll0147 gene knockout and overexpression Synechocystis sp. and wild-type Synechocystis sp. The specific operation steps are as follows: take 50mlOD 730 =1.8 cyanobacteria, 4°C, 5000rpm centrifugation for 10min to collect algae cells, add 0.4mL neutral phenol to 0.4mL BG-11 liquid medium, then add an appropriate amount of glass beads (purchased from sigma company) with a diameter of about 0.17mm to the glass There is 0.5 mL of suspension above the bead interface. Vibrate with a vortex shaker at the maximum speed for 1min, centrifuge at 11900rpm for 10min at 4°C, take the supernatant to ...
PUM
Login to view more 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