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A kind of Clostridium anaerobic xylan-inducible promoter and its application

An anaerobic clostridium and promoter technology, applied in the field of genetic engineering, can solve cumbersome problems and achieve the effect of strong versatility and simple method

Active Publication Date: 2020-04-24
QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, the detection of promoter activity generally depends on the enzymatic reaction system, and the existing problems are more prominent, (1) cumbersome, and the enzymatic reaction generally depends on the utilization of the substrate and the detection of the signal; (2) there are strains selectivity, such as the background level expression of enzymes in some cells

Method used

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  • A kind of Clostridium anaerobic xylan-inducible promoter and its application
  • A kind of Clostridium anaerobic xylan-inducible promoter and its application
  • A kind of Clostridium anaerobic xylan-inducible promoter and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Acquisition of promoter and Clostridium cellulolyticum transformants:

[0047] 1) Acquisition of the promoter

[0048] Design primers to F:AA CTGCAG TCAAGATATTTTATAA CTAAAGAT; R:CG ACGCGT CATGTTCTCAAATCCTCC, as a pair of primers, was cloned into the promoter (ie, p3398) fragment of Ccel_3398 (eg, the fragment shown in SEQ ID NO: 1) by means of PCR using the genome of Clostridium cellulolyticum as a template. The functional region of this promoter includes: the SD sequence (ribosome binding site) upstream of the translation initiation codon ATG - AGGAGG, the transcription initiation site is G, the TATA box in the -10 region - TATAAAT, and the -35 region - TTGGAAAT, and upstream regulatory regions.

[0049] 2) Obtaining recombinant plasmids

[0050] Using the restriction endonucleases PstI and MluI to carry out the double digestion reaction of the above obtained p1133 fragments in a 37 degree water bath to obtain the p1133 fragments with cohesive ends;

[0051]At t...

Embodiment 2

[0069] Establishment of reporter system based on anaerobic fluorescent protein

[0070] Generally speaking, the study of promoter characteristics should be based on a simple, reliable and intuitive reporting system, so that the activity of the promoter can be displayed. At present, most reporter systems rely on enzymatic reactions, but the operation is basically very cumbersome, and it is still difficult to achieve high throughput. Although GFP (Green Fluorescent Protein)-based promoter screening has achieved high-throughput, such as FACS fluorescence-excited cell screening, because GFP is oxygen-dependent, it cannot be applied to anaerobic cells. However, the present invention uses anaerobic fluorescent protein as the reporter system, which avoids the shortage of GFP well. The present invention further investigates the reliability of the anaerobic fluorescent protein-based reporter system.

[0071] 1) Cell culture

[0072] Two kinds of cell culture are involved here, one i...

Embodiment 3

[0085] Example 3——Application of xylan-inducible promoter

[0086] The functional identification of the above-mentioned obtained promoters is mainly based on the differences in the promoter activities exhibited by the promoters under different carbon source conditions. Specific maintenance:

[0087] 1) Cell culture

[0088] Inoculate the Clostridium cellulolyticum transformants containing the recombinant vector pXL007 obtained in the above examples into DCB-1 medium containing 20 μg / ml erythromycin, in a Hengate roller tube at a constant temperature of ~35 degrees, and anaerobically culture at 0.0005 % resazurin was used as an oxygen indicator, and cultured to the middle and late logarithmic stages. Bacterial inoculation and transfer were performed using 1ml syringes.

[0089] 2) Identification of inducible promoters

[0090]The Clostridium cellulolyticum transformants containing the recombinant vector pXL007 and the control cells (cells containing the pth1 promoter, ie ce...

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Abstract

The invention relates to the field of gene engineering, and specifically relates to an anaerobic clostridium xylan induction type promoter and applications thereof. The promoter is described as follows: (a) the sequence is the DNA fragment shown in the sequence table 1; (b) the promoter comprises a DNA fragment, which is at least 90% of homology with the nucleotide sequence defined in (a) and has a promoter function; and (c) the promoter comprises a DNA fragment, which is hybridized with the nucleotide sequence defined in (a) or (b) under strict conditions and has a promoter function. The induction type promoter in anaerobic clostridium cellulolyticum is screened by a method of mainly taking riboflavin mononucleotide dependent fluorescent protein (FbFP) as the reporting system. FbFP is cultured in culture mediums taking five different carbon sources as the substrates so as to obtain different expression amount, the expression amount is lowest (the carbon source is cellobiose), and the expression is the highest (the carbon source is xylan). The provided induction type promoter has an important meaning for the controllable genetic expression and genetic modification of gram positive bacteria. At the same time, the genetic elements of synthetic biology are enriched.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an anaerobic clostridium xylan-inducible promoter and its application. Background technique [0002] Anaerobic Clostridium plays a very important role in the degradation of lignocellulose and contributes to the renewable link of energy. Clostridium cellulolyticum is a class of mesothermic anaerobic Clostridium capable of utilizing multiple substrates (cellodextrin, cellobiose, glucose, xylose, xylan, arabinose, etc.), and is a class of potential application A strain fermented in one step with CBP. With the improvement of its genome and transcriptome, Clostridium cellulolyticum has increasingly become a model organism for the study of cellulose degradation. However, the process of its genetic modification is the only way for it to be applied in industrial production, so it is very necessary to establish a set of efficient and simple controllable promoter system in Clostridium ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/74C12N1/21
Inventor 许成钢滕琳徐健
Owner QINGDAO INST OF BIOENERGY & BIOPROCESS TECH CHINESE ACADEMY OF SCI