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A strain of Enterobacter cloacae FY-0701, construction method and applications thereof

A technology of FY-0701, Enterobacter cloacae, applied in the field of genetic engineering, can solve problems such as affecting the ability of deep profile control and affecting the efficiency of bacterial migration in oil reservoirs

Active Publication Date: 2019-01-01
NANKAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Enterobactersp.FY-07 can produce a large amount of BC when various common carbon sources are used as the only carbon source, which will affect the migration efficiency of bacteria in the oil reservoir, thereby affecting the ability of deep profile control

Method used

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  • A strain of Enterobacter cloacae FY-0701, construction method and applications thereof
  • A strain of Enterobacter cloacae FY-0701, construction method and applications thereof
  • A strain of Enterobacter cloacae FY-0701, construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0136] Selection of target sites for genetically engineered strain construction

[0137] The present invention aims to construct a bacterial strain that can produce bacterial cellulose when carbohydrates are used as carbon sources, but not produce bacterial cellulose when non-sugars are used as carbon sources. Therefore, RNA extraction and reverse transcription quantitative PCR were used to analyze the similarities and differences in the expression of the key gene fructose-1,6-2 phosphorylase in the gluconeogenesis pathway of Enterobactersp.FY-07 when glycerol or glucose was used as the only carbon source. At the late stage of logarithmic growth, the Enterobactersp.FY-07 bacterial cells cultured with glucose or glycerol as the sole carbon source were collected by centrifugation at 4°C and 5000rpm for 10min, and quickly placed in liquid nitrogen. The growth curve under carbon source conditions is as follows figure 1 shown. Bacterial total RNA was extracted with Purezol bindin...

Embodiment 2

[0194] Construction of gene knockout vector

[0195] Using the genome of Enterobacter cloacae FY-07 as a template, use the Fpg1u, Fpg1l primer pair and Fpg3u, Fpg3l primer pair to amplify the upstream and downstream homology arms of the target knockout gene, and use the Fpg2u, Fpg2l primer pair to amplify the green fluorescent protein gene. Using the Fpgnu and Fgnl primer pair and the method of overlapping PCR, the upstream and downstream homology arms and the green fluorescent protein gene were connected into a single PCR product Fpg. The Fpg fragment was double-digested with EcoRI and XbaI, and pTSK1 was double-digested with the same enzymes. The two digested products were ligated with T4DNALigase, transformed into E.coli DH5α competent cells, and the gene knockout vector pTSFPG was obtained. The primers used in this example are shown in Table 4.

[0196] Table 4 Gene knockout vectors and primer information during the construction of gene knockout strains

[0197]

[0...

Embodiment 3

[0225] Construction of genetically engineered strains

[0226] The genetic engineering vector was transferred into Enterobacter cloacae FY-07 by conjugative transfer. Correct transformants were resistant to carbenicillin and tetracycline and were verified by PCR with primers designed using the pTSK1 backbone as a template. Transformants that were screened correctly were cultured overnight at 42°C under the condition of no selective pressure. Overnight cultures were diluted onto LB plates containing tetracycline resistance. The primer pair Fpgk1u and Fpgk1l and the primer pair Fpgk2u and Fpgk2l were used to perform colony PCR verification on single colonies grown on LB plates to screen for single exchange transformants. Single-crossover transformants were passaged under non-selective stress conditions and diluted onto LB plates containing 10% sucrose. The primer pair Fpgk1u and Fpgk1l and the primers Fpgk2u and Fpgk2l were used again to perform colony PCR verification and sc...

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Abstract

The invention provides a strain of Enterobacter cloacae FY-0701, a construction method and applications thereof, and belongs to the technical field of genetic engineering, wherein the preservation number of the Enterobacter cloacae FY-0701 is CGMCC No.15619. The Enterobacter cloacae FY-0701 of the present invention can achieve the deep profile control effect.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a strain of Enterobacter cloacae FY-0701 and its construction method and application. Background technique [0002] In recent years, tertiary recovery technology has been applied to enhance crude oil production (Green and Willhite, 1998). Microbial enhanced oil recovery (MEOR) technology has many advantages such as environmental friendliness, low cost and high efficiency, high biodegradability and low Toxicity is considered an important tertiary mining technique (Ghojavand et al., 2012; Kobayashi et al., 2011; Lazar et al., 2007; Makkar et al., 2011). MEOR technology uses microorganisms and / or their metabolites such as organic acids, gases, biosurfactants and polymers to extend the life of the reservoir by injecting nutrients into the well and cultivating microorganisms (Sen, 2008; Sun et al., 2011) . [0003] Heterogeneity is one of the main reasons for low oil rec...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/74C12N15/90C09K8/582C09K8/514C12R1/01
CPCC09K8/514C09K8/582C12N9/16C12N15/74C12N15/902C12Y301/03011
Inventor 马挺纪凯华李国强高歌
Owner NANKAI UNIV
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