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Function identification of promoter of source sequence of gluconacetobacter xylinum and application of promoter in promotion of synthesis of bacterial cellulose

A technology of gluconacetobacter and bacterial cellulose, applied in the biological field, can solve problems such as unclear functions, high cost of inducers, and inability to open constitutive promoters, so as to promote the synthesis of d bacterial cellulose, increase cognition, Effects of high promoter activity

Pending Publication Date: 2022-07-05
TIANJIN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Due to the complexity and diversity of the fermentation process, there are many problems in the application of the current constitutive promoters and inducible promoters, such as the high cost of inducers, the constitutive promoters cannot be turned on under specific conditions, etc., while the xylose The promoter derived from Acetobacter does not have this kind of problem, and this kind of promoter can be well used for the genetic engineering of the strain itself, and can accurately turn on the expression of the gene
The function of the promoter of Gluconacetobacter xylinum-derived sequence is still unclear

Method used

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  • Function identification of promoter of source sequence of gluconacetobacter xylinum and application of promoter in promotion of synthesis of bacterial cellulose
  • Function identification of promoter of source sequence of gluconacetobacter xylinum and application of promoter in promotion of synthesis of bacterial cellulose
  • Function identification of promoter of source sequence of gluconacetobacter xylinum and application of promoter in promotion of synthesis of bacterial cellulose

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Experimental program
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Effect test

Embodiment 1

[0048] The promoter strength of the Acetobacter xylinum derived sequence characterizes the construction of the plasmid.

[0049] In order to characterize the expression strength of the promoter of the sequence derived from Acetobacter xylinum 01095 Express the red fluorescent protein gene. First, a backbone plasmid pBRS was constructed for subsequent studies. The backbone plasmid is based on the pBla vector with the promoter, ribosome binding site (RBS) and terminator removed, and is composed of BamHI restriction site, RBS, mRFP and TrrnB terminator. According to the published genome sequence of Acetobacter xylinum CGMCC 2955 and P 01095 Promoter annotation information, design primers P 01095 -F / R, using the CGMCC 2955 genome as a template to obtain P by PCR amplification 01095 promoter fragment. Use BamHI to digest the vector pRBS to obtain a linear vector, and then clone and connect the above two fragments through Novozan's one-step recombination kit to obtain the pRBS-...

Embodiment 2

[0053] Construction and strength characterization of promoter-screening plasmids for sequences derived from Acetobacter xylinum.

[0054] The red fluorescent protein gene mRFP was used as the reporter gene, and the control was P on the commonly used pBla plasmid. bla Promoter, examine expression element P 01095 Fluorescent protein expression characteristics, and its fluorescence intensity at 96h was determined.

[0055] 1. Determination of fluorescent protein expression intensity

[0056] The fluorescent protein expression assay method is as follows: pick a single colony from the solid medium plate with an inoculation loop and inoculate it in 5 mL of liquid medium at 180 r / min at 30 °C for 24 h. Transfer 1 mL of fermentation broth to 75 mL of seed medium containing 4‰(v / v) cellulase (Celluclast 1.5L, Novozymes), and cultivate to OD at 180 r / min at 30 °C. 600 is 0.5 to 0.6. Take 750μl of seed solution and transfer it to 75mL liquid medium (initial pH 6.0, initial glucose co...

Embodiment 3

[0063] Sequences with strong promoter functions derived from Acetobacter xylinum are used in the synthesis of bacterial cellulose in Acetobacter xylinum.

[0064] 1. Construction of a recombinant vector of a sequence with strong promoter function derived from Gluconobacter xylinum in Acetobacter xylinum.

[0065] According to the reported genome sequence of Acetobacter xylinum CGMCC 2955, the genome of CGMCC2955 was used as the template, and P 01095 -pfkA-F / R,P bla - pfkA-F / R is the primer, PCR amplification of the target gene pfkA, after the fragment is recovered, cloned and ligated by Novozan's one-step recombination kit to obtain the recombinant vector pRBS-P 01095 -pfkA and pRBS-P bla -pfkA. The primer sequences used above are shown in Table 3.

[0066] table 3

[0067]

[0068] 2. Construction of bacterial cellulose-producing bacteria having a sequence with strong promoter function derived from Acetobacter xylinum.

[0069] The recombinant vector pRBS-P construct...

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Abstract

The invention discloses a sequence which is derived from gluconacetobacter xylinum and has a promoter function and application of the sequence in promoting synthesis of bacterial cellulose. The sequence has higher promoter activity than a pBla plasmid promoter. Therefore, the strain can be used for enhancing the expression of a target gene, for example, the expression intensity of phosphofructokinase pfkA is enhanced by operably connecting the strain with a phosphofructokinase gene, so that the bacterial cellulose production efficiency of the strain is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the identification of a promoter function of a sequence derived from Acetobacter xylinum and its application in promoting bacterial cellulose synthesis. Background technique [0002] Bacterial cellulose is a nanoscale biopolymer synthesized by bacteria. It has excellent qualities such as high crystallinity, high degree of polymerization, high tensile strength, high water holding capacity, biocompatibility and degradability, making it widely used in biomedicine, food, textile, functional film, cosmetics and audio equipment and other industries. Cellulose-producing bacterial strains include Agrobacterium, Rhizobium, Pseudomonas, and Acetobacter. Among them, Acetobacter xylinum is the most widely studied bacterial cellulose-producing strain due to its ability to produce high bacterial cellulose. With the continuous development of biotechnology, reports of metabolic engine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/74C12N15/54C12P19/04C12R1/01
CPCC07K14/195C12N15/74C12N9/1205C12P19/04C12Y207/01011
Inventor 辛波钟成王旭彭昭君李文超谢燕燕贾士儒
Owner TIANJIN UNIV OF SCI & TECH
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