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Corynebacterium glutamicum artificial promoter library

A Corynebacterium glutamicum, promoter technology, applied in the field of synthetic biology and metabolic engineering, can solve problems such as limiting the application of Corynebacterium glutamicum

Pending Publication Date: 2019-10-11
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, in Corynebacterium glutamicum, there are still few studies on gene expression control elements, especially the discovery of biological elements with simple composition, precise regulation, and plug-and-play has not been reported, which greatly limits the ability of glutamine Further application of Corynebacterium acid

Method used

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  • Corynebacterium glutamicum artificial promoter library

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Experimental program
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Effect test

Embodiment 1

[0027] The construction of embodiment 1 Corynebacterium glutamicum artificial promoter element library

[0028] The strategy for the construction of the artificial promoter element of Corynebacterium glutamicum is based on the integration of σ A and σ B The core region of the dependent promoter, characterized by its core regions -35 (NNGNCN) and -10 (NNTANANT), and a ribosome binding site (AAAGGANNNNN). In order to construct the Corynebacterium glutamicum artificial promoter element library, the designed Corynebacterium glutamicum artificial promoter element, the upstream primer of the reporter gene and the Bsa I restriction site were integrated to design random primers with a length of 120 bases ( figure 1 ).

[0029] By means of PCR, the green fluorescent protein is used as a template to amplify the nucleotide fragment I containing the BsaI restriction site, the artificial promoter element of Corynebacterium glutamicum and the reporter gene. The primer sequences are:

[...

Embodiment 2

[0042] The high-throughput screening of embodiment 2 Corynebacterium glutamicum artificial promoter element library

[0043] The Corynebacterium glutamicum constructed above containing the artificial promoter element library was cultured at 32° C. for 48 h. Use 1~5mLddH 2 O washed the cells, transferred to 100mL LBHIS liquid medium, and cultured at 32°C and 200rpm for 2h. Take 1mL of bacterial solution, and use sterilized PBS buffer solution (NaCl 137mmol / L, KCl 2.7mmol / L, NaCl 2 HPO 4 10mmol / L, KH 2 PO 4 2mmol / L), washed 3 times and resuspended. Dilute the bacterial solution to OD 600 = 0.1 to 0.5. The obtained bacteria were sorted by flow cytometry using MoFlo XDP FlowCytometry Sorter (Beckman Coulter, USA). Select the fluorescence channel as the excitation wavelength 488nm, and the emission wavelength as the 530 / 40 channel. Select the flow cytometry purification mode to increase the proportion of positive clones in the screened library. The top 0.1-1% cells were ...

Embodiment 3

[0046] The characterization of embodiment 3 Corynebacterium glutamicum artificial promoter element

[0047] The Corynebacterium glutamicum artificial promoter element obtained above is characterized, and its steps are as follows:

[0048] Among the selected 760 recombinant strains, they were divided into three groups according to the intensity of fluorescence. A promoter with a fluorescence intensity greater than 8000 a.u. is a high-expression promoter, a promoter with a fluorescence intensity between 3000 and 6000 a.u. is a medium-intensity promoter, and a fluorescence intensity less than 2000 a.u. is a low-intensity promoter. Ten promoters were randomly selected from each of the three different-strength promoter groups for further analysis.

[0049] The 30 promoters were inoculated in LBHIS medium containing chloramphenicol, and cultured in shake flasks for 16 hours. Utilize multifunctional microplate reader to carry out fluorescence intensity detection then, excitation wa...

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Abstract

The invention discloses a library of artificial promoter regulatory elements with different expression intensities in corynebacterium glutamicum. Firstly, according to the core region of promoters ofcorynebacterium glutamicum housekeeping genes and the conserved sequences of the -10 region and the -35 region, the corynebacterium glutamicum artificial promoter regulatory element library is designed and built. Each regulatory element comprises a promoter region and a ribosome binding site region, and a promoter upstream region exists. Subsequently, the artificial promoter regulatory element library is subjected to a three-step screening method of flow cytometry screening, plate screening and 96-well plate screening, and the artificial promoter regulatory element library with with differentexpression intensities and wide control range is obtained.

Description

technical field [0001] The invention belongs to the technical field of synthetic biology and metabolic engineering, and in particular relates to a corynebacterium glutamicum artificial promoter regulatory element library and a construction method thereof. Background technique [0002] Corynebacterium glutamicum is a food-safe Gram-positive strain isolated from soil, which is widely used in the production of amino acids and other products. In recent years, with the development of synthetic biology, artificial cell factories based on Corynebacterium glutamicum have attracted more and more attention for the synthesis of various bioenergy and fine chemical products. However, due to the complexity of the intracellular metabolic network, during the construction of metabolic pathways and the modification of targeted metabolic nodes, the inappropriate expression of gene expression usually disturbs the metabolism of host cells, causing an imbalance in metabolic flux, and then Affect...

Claims

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Application Information

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IPC IPC(8): C12N15/113C40B50/06C40B40/06C12N15/77
CPCC12N15/113C12N15/77C40B50/06C40B40/06C12N2830/34
Inventor 刘君魏亮周威徐宁
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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