A kind of engineering bacteria producing coniferyl alcohol, its construction method and the application of producing coniferyl alcohol
A construction method and technology for engineering bacteria, which are applied in the directions of microorganism-based methods, biochemical equipment and methods, bacteria, etc., to achieve the effects of being beneficial to industrial production, solving source problems, and reducing production costs.
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Embodiment 1
[0030]Example 1 Design of TAL, COMT, 4CL and CCR genes
[0031]Phanerochaete chrysosporium (Phanerochaete chrysosporium) tyrosine deaminase TAL, Arabidopsis thaliana (Arabidopsis thaliana) O-methyltransferase COMT, coumadin-4-CoA ligase 4CL, cinnamoyl Coenzyme A reductase CCR, using JCAT online codon optimization software (http: / / www.jcat.de) combined with OPTIMIZER codon optimization tool (http: / / genomes.urv.es / OPTIMIZER / ), use E. coli to pair the code The preference of the child is optimized, and the full-length TAL, COMT, 4CL and CCR genes are designed. The nucleotide sequences of the genes TAL, COMT, 4CL and CCR are shown as SEQ ID No. 05, SEQ ID No. 06 and SEQ in the sequence table. ID No.07 and SEQ ID No.08 are shown.
Embodiment 2
[0032]Example 2 The lambda-red homologous recombination method integrates the T7 RNA polymerase gene into the chromosome of the chassis strain SyBE-002447.
[0033]The detailed steps of the construction process of the chassis strain integrating the T7 RNA polymerase gene into the chassis strain SyBE-002447 chromosome are as follows:
[0034]1. Using the E. coli BL21 (DE3) genome as a template, the T7 RNA polymerase gene was cloned using Fastpfu enzyme for PCR reaction.
[0035]2. Use overlap extension PCR to prepare chloramphenicol-resistant T7 RNA polymerase fragments.
[0036]3. Introduce the pKD46 plasmid into the strain SyBE-002447 to obtain SyBE-002447 / pKD46. Inoculate the activated strain SyBE-002447 / pKD46 in 10ml LB liquid medium at 30℃, 200rpm, and cultivate to OD600Is 0.4-0.6. Add L-arabinose to a final concentration of 10mM, and continue to incubate for 3h. Centrifuge at 4000 rpm for 8 min at 4°C to collect the cells. The supernatant was discarded, and ice-cold 10% glycerol was added ...
Embodiment 3
[0039]Example 3 Construction of recombinant expression vector pTHM
[0040]Using the E. coli genome as a template, the Fastpfu enzyme was used for PCR reaction to obtain the hpaBC gene with KpnI and XhoI restriction sites on both ends (the nucleotide sequence uses SEQ ID No. 01). The PCR reaction was used to make the two ends of the TAL gene fragment obtained by chemical total synthesis have HindIII and BamHI restriction sites respectively; the two ends of the COMT gene fragment were equipped with HindIII and NotI restriction sites.
[0041]The full chemical synthesis expression vector backbone pBPE001, its sequence is shown in SEQ ID No.09 in the sequence table.
[0042]Use FastDigest endonuclease HindIII and BamHI to digest the TAL gene and pBPE001. The reaction system is: 5μL 10*FD buffer, 2.5μL HindIII, 2.5μLBamHI, 30μL TAL gene fragment and 10μL ultrapure water. The reaction conditions are: 37°C, 1h. Use PCR purification kit to recover the digestion system, add 250μL Binding Buffer solu...
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