A kind of strong promoter and its plasmid carrier and application
A plasmid vector, strong promoter technology, applied in the biological field, can solve the problem of limited number of efficient promoters
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0021] Example 1: Construction of a promoter-containing plasmid vector
[0022] 1. Preparation of vector containing reporter gene
[0023] Utilize the primers gfp-EcoRI-F and gfp-XhoI-R in Table 1, and use the ECE164 plasmid as a template to amplify by PCR and introduce EcoRI and XhoI restriction sites to obtain the gfp fragment of the green fluorescent protein gene. After verification by electrophoresis, DpnI enzymatic treatment, and electrophoresis gel recovery, the purified gfp fragment was obtained. The purified gfp fragment and pBBR1MCS2 plasmid were double-digested with EcoRI and XhoI respectively, and the two double-digested products were ligated overnight at 4°C with T4 ligase. The ligation product was transformed into Escherichia coli DH5α, spread on the LB solid plate containing 50mg / L kanamycin, cultured for 16 hours, then carried out colony PCR detection, and sent to Jinweizhi for sequencing. After the sequence was correct, the obtained positive bacteria were name...
Embodiment 2
[0029] Embodiment 2: The activity determination of promoter P21 in different strains
[0030] 1. Transformation—three-parent transformation method
[0031] Taking S. meliloti as an example, the plasmid pBBR-P21-gfp in Example 1 was transferred into S. meliloti according to the three-parent method to obtain S. meliloti: SM / pBBR-P21-gfp. Specific steps are as follows:
[0032] (1) Inoculate the newly activated Sinorhizobium meliloti CGMCC NO.9638, Escherichia coli (containing the corresponding plasmid) and the helper vector MT616, and shake culture in the incubator at 30°C and 37°C respectively until the OD value is about 1.0;
[0033] (2) Under aseptic conditions, transfer 500 μL each of Sinorhizobium meliloti CGMCC NO.9638, MT616 and Escherichia coli to 1.5 mL sterile EP tubes and centrifuge at 12,000 rpm for 1 min at 4°C.
[0034] (3) Discard the supernatant under aseptic conditions, and suspend the precipitate with 1 mL of 0.85% sterile saline.
[0035] (4) Centrifuge aga...
PUM
Abstract
Description
Claims
Application Information
- 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, Popular Technical Reports.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com