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Nucleic acid construct comprising 5' utr stem-loop for in vitro and in vivo gene expression

A technology of nucleic acid constructs and constructs, applied in the direction of using vectors to introduce foreign genetic material, genetic engineering, biochemical equipment and methods, etc.

Pending Publication Date: 2022-02-01
GLYCOM AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

It has been proposed that the global transcriptional regulator cAMP-CRP or CRP formed upon glucose limitation regulates a minimum of 378 promoters in bacterial cells (Shimada T. et al., PloSOne 6(6):e20081, (2011)), however, no Data indicating which promoters are suitable to drive high-yield genome-based, stable and controllable production of recombinant biomolecules in an industrial setting

Method used

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  • Nucleic acid construct comprising 5' utr stem-loop for in vitro and in vivo gene expression
  • Nucleic acid construct comprising 5' utr stem-loop for in vitro and in vivo gene expression
  • Nucleic acid construct comprising 5' utr stem-loop for in vitro and in vivo gene expression

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Embodiment approach

[0118] The following are some selected but non-limiting embodiments of the invention.

[0119] In one embodiment, the present invention relates to an isolated nucleic acid sequence identified as SEQ ID NO:1. In another embodiment, the present invention relates to a variant of SEQ ID NO:1, wherein said variant has at least 80% sequence identity to SEQ ID NO:1.

[0120] Preferably, the isolated nucleic acid sequence identified as SEQ ID NO: 1 is comprised in a nucleic acid construct.

[0121] In one embodiment, the construct comprises a promoter DNA sequence operably linked to a continuous synthetic DNA sequence (i),

[0122] in

[0123] a) the DNA sequence (i) has a length of at least 23 nucleobases and comprises SEQ ID NO: 1, or a variant thereof; wherein said variant has at least 80% sequence identity to SEQ ID NO: 1;

[0124] b) A promoter is an isolated DNA sequence that contains a single binding site for cyclic AMP receptor protein (CRP) centered at about -41 after the ...

Embodiment 1

[0181] Example 1 - Regulation of gene expression by replacing part of the 5'UTR with synthetic DNA comprising 54UTR-glpF in the expression elements PgatY_org and PmglB_org respectively

[0182] Four DNA fragments containing various promoter elements were cloned using a promoter-probe plasmid containing the promoterless lacZ gene. Expression levels of lacZ were determined following integration of the promoter-lacZ element into chromosomal DNA in a single copy following fusion of the promoter element to lacZ. The deletion of AlacZM15 in the lacZ gene in E. coli MDO renders it unable to produce active β-galactosidase and was therefore used as the strain background in the screening. Two recombinant nucleic acid sequences comprising genomic promoter sequences derived from the operons gatYZABCDR and mglBAC were fused to a promoterless lacZ reporter gene and inserted into chromosomal DNA in a single copy. Measure the expression level of the cloned fragment ( figure 1 , IOU). The e...

Embodiment 2

[0183] Example 2 - Regulation of Expression of Recombinant Nucleic Acid Sequences Using Synthetic PmglB Expression Elements

[0184] We have previously demonstrated the effect of modification of the 16UTR / Rec UTR (SEQ ID NO:3) sequence on gene expression from PglpF_70UTR and PglpT_70UTR constructs containing this sequence (PCT / IB2018 / 060355). Here we confirm that the 16UTR variant described in PCT / IB2018 / 060355 in combination with 54UTR-glpF has a similar effect on the expression of the lacZ gene from a construct comprising PmglB. Alteration of the 16 nucleotide DNA fragment of mglB-5' URT located just upstream of the mglB translation initiation site with a synthetic DNA fragment (16UTR, SEQ ID NO:3) resulted in a 2-fold increase in expression. Replacement of the entire mglB 5'UTR region located between the transcription start site and the translation start codon with the glpF-70UTR sequence yields PmglB_70UTR (SEQ ID NO:25) with increased expression levels compared to the ori...

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Abstract

The present invention relates to the field of recombinant production of biological molecules in host cells. The invention provides nucleic acid constructs that allow to modify expression of a desired gene using both in vitro and in vivo gene expression systems with optimized stem-loop structures in the 5' UTR of said genes. The constructs can advantageously be used to produce a variety of biological molecules recombinantly in industrial scales, e.g. human milk oligosaccharides (HMOs).

Description

technical field [0001] The present invention relates to the field of recombinant production of biomolecules in host cells. The present invention provides nucleic acid constructs that allow the use of in vitro and in vivo gene expression systems to modify the expression of a desired gene. The constructs can be advantageously used for the recombinant production of various biomolecules such as human milk oligosaccharides (HMOs) on an industrial scale. Background technique [0002] The commercial importance of recombinant microorganisms to produce biomolecules is increasing. Currently, expression systems carried by plasmids are mainly used to produce recombinant proteins in bacterial hosts, especially Escherichia coli (E.coli). These systems have gained wide acceptance due to the high gene dosage they offer and the ease of handling available cloning protocols. However, there are also a number of disadvantages to using plasmid-based expression systems, especially at the manufa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/70
CPCC12N15/70C12N15/67C12N15/113C12N2310/531
Inventor M·彼得森
Owner GLYCOM AS