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Vector for knocking out L-lactic dehydrogenase 1 gene and construction method of vector

A technology of lactate dehydrogenase and construction method, which is applied in the field of carrier and construction for knocking out L-lactate dehydrogenase 1 gene, can solve the problems of time-consuming, difficult to succeed and low efficiency of enzymatic digestion and connection, and achieve splicing Effects of improved reaction conditions, reduced use times, and cost and time savings

Inactive Publication Date: 2015-06-03
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technique allows researcher to isolate specific types of microorganisms called Listeria monocytora (LM) while also creating their own modified versions or hybrid strains containing different genes involved in metabolism. By combining these modifications together into one new clone, it becomes possible to study how certain parts work better than others when combined alone. These techniques allow scientists to make more precise experiments at an affordable price compared to traditional methods such as cloning and reverse transcriptases.

Problems solved by technology

This patents describes how expensive materials like dluxylonium (dioxygen) have become necessary due to their importance in producing various products from biopolymer resin compositions containing these compounds. However, current techniques require either expensive starting materials or complicated processes involving multiple steps which result in lower yields compared to desired product levels. There is thus a technical problem related to developing efficient ways to make highly optically purified polymer molecules without introducing new components during construction process.

Method used

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  • Vector for knocking out L-lactic dehydrogenase 1 gene and construction method of vector
  • Vector for knocking out L-lactic dehydrogenase 1 gene and construction method of vector
  • Vector for knocking out L-lactic dehydrogenase 1 gene and construction method of vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1 Preparation of Lactobacillus plantarum DM9010 genome and extraction of recombinant plasmid. Including the following steps:

[0032] (1) Centrifuge 1.5ml of the overnight cultured Lactobacillus plantarum solution and collect the precipitate, add 0.6mL of 20mg / ml lysozyme, invert and mix for 5-10 times, then place it at 37°C for 40min;

[0033] (2) Centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant carefully.

[0034] (3) Add 150 μL SP Buffer (containing RNase A1) to fully suspend the bacterial pellet.

[0035] (4) Add 20 μL of Lysozyme solution, mix evenly and let stand at room temperature for 5 minutes.

[0036] (5) Add 30 μL of EDTA Buffer, mix evenly and let stand at room temperature for 5 minutes.

[0037] (6) Add 200 μL of Solution A, shake vigorously and keep warm at 65°C for 10 minutes.

[0038] (7) Add 400 μL of Solution B and shake vigorously for 15 seconds.

[0039] (8) Add 650 μL of Solution C and mix evenly by inve...

Embodiment 2

[0052] Example 2 The upstream and downstream fragment primer design and PCR amplification of the Lactobacillus plantarum DM9010L-lactate dehydrogenase 1 gene, the specific operations are as follows:

[0053] (1) According to the reported gene sequence encoding L-lactate dehydrogenase 1, BLAST was performed on Lactobacillus plantarum WCFS1, ST-Ⅲ, JDM1, ZJ316, P8, and 16 whose genomes had been sequenced in NCBI to find L-lactate The location of the gene encoding dehydrogenase 1 and its upstream and downstream DNA sequence of about 1000 bp, then determine the conserved sequence to design primers and introduce restriction sites and 15 bp overlapping fragments.

[0054] (2) PCR amplifies upstream DNA fragment 978bp, downstream fragment 870bp, determines the reaction system of 50 μ l as follows: Lactobacillus plantarum DNA template 5 μ l, 5×PCR PrimerSTAR, Buffer (Mg 2+ plus) 10 μl, dNTP Mixture 4 μl, each primer 1 μl, PrimerSTAR HS DNA Polymerase 0.5 μl, add water to 50 μl. The re...

Embodiment 3

[0055] Example 3 Condition optimization of overlap extension PCR ligation of upstream and downstream DNA fragments. The specific operation is as follows:

[0056] (1) Step-by-step overlap extension PCR reaction process is divided into three steps: (a) Use high-fidelity DNA polymerase to amplify the upstream and downstream fragments respectively, and cut the gel for recovery. (b) Overlap extension of the upstream and downstream DNA fragments, high temperature denaturation during the reaction process to make the upstream and downstream DNA fragments into single strands, and then annealing to pair the overlapping regions (30bp) of the upstream and downstream DNA fragments, and extend each other as templates. The reaction system is as follows: 5×PCR PrimerSTAR, Buffer (Mg 2+ plus) 10 μl, dNTP Mixture 4 μl, upstream DNA fragment 2 μl (about 20 ng), downstream DNA fragment 2 μl (about 20 ng), PrimerSTAR HS DNA Polymerase 0.5 μl, add water to 50 μl. Extension reaction conditions: 9...

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Abstract

The invention discloses a vector for knocking out an L-lactic dehydrogenase 1 gene and a construction method of the vector. The construction method comprises the following steps: completely splicing upstream and downstream DNA fragments of a gene which does not contain an L-lactic dehydrogenase encoding gene by using an overlapping extension process, also cloning the spliced fragments to a sub-vector pMD18-T and transforming the spliced fragments to escherichia coli DH5alpha competent cells, screening constructed sub-cloning vectors by virtue of blue-white spots, performing double enzyme digestion to obtain a target strip, then connecting the target strip to a pG+host9 plasmid, and performing bacterial colony PCR and double enzyme digestion identification to successfully obtain a recombinant vector. According to the vector and the construction method thereof disclosed by the invention, a recombinant plasmid of which the L-lactic dehydrogenase encoding genes are knocked out is constructed by using an overlapping extension PCR process, so that excessive base sequences introduced by enzyme digestion and connection can be avoided; and the recombinant vector disclosed by the invention can be used for producing D-lactic acid with high optical purity.

Description

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Claims

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Application Information

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Owner SOUTH CHINA UNIV OF TECH
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