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Artificial promoter libraries for selected organisms and promoters derived from such libraries

A technology of promoters, organisms, applied in the direction of using vectors to introduce foreign genetic material, DNA preparation, DNA/RNA fragments, etc., which can solve problems such as randomization of spacers

Inactive Publication Date: 2006-03-01
彼得·鲁戴尔·简森
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

To date, no one has attempted to randomize the spacer while keeping the length of the consensus sequence and the spacer relatively constant

Method used

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  • Artificial promoter libraries for selected organisms and promoters derived from such libraries
  • Artificial promoter libraries for selected organisms and promoters derived from such libraries
  • Artificial promoter libraries for selected organisms and promoters derived from such libraries

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Design of degenerate oligonucleotides for a Lactococcus lactis promoter library

[0060] According to the reference (see review by de Vos & Simons, 1994), strong promoters in Lactococcus lactis tend to share the following nucleotide sequences (numbers refer to positions relative to the transcription start site, which The position numbers of the sites are +1): -12 to -7: TATAAT; -15 to -14: TG; -35 to -30: TTGACA. The interval between -10 and -35 appears to be 17 nucleotides. However, a more careful comparison of the published L. lactis promoter sequences revealed that the nucleotides are also more or less conserved at many positions other than those mentioned above. Some such positions are: -1: A; -3: A or T (=W); -6: A; -13: A or G (=R); -40 to -36: TATTC. In addition, Nilsson and Johansen (1994, BBA) pointed out two motifs, +1 to +8: GTACTGTT and -44 to -41: AGTT, which are relatively strong promoters in Lactococcus lactis (for transfer RNA and promoters of ribosom...

Embodiment 2

[0100] Design of degenerate oligonucleotides for a temperature-regulated Lactococcus lactis promoter library

[0101] This example illustrates the development of a temperature-regulated promoter library for Lactococcus lactis. A regulatory element containing an 8 base pair inverted repeat, which has been shown to be involved in the heat shock response of Lactococcus lactis, was inserted a few base pairs upstream of the -35 sequence. This regulatory element is a minimum of 27 base pairs:

[0102] 5′-TTAGCACTCNNNNNNNNNGAGTGCTAA-3′

[0103] IR Spacer IR

[0104] It contains 9 bp (or longer) inverted repeats (IRs) separated by 9 (or less) base pairs. It should therefore be possible to combine this inverted repeat sequence with a method for obtaining constitutive promoters of different strengths, thereby obtaining a series of promoters with various basic activities, which can be induced several-fold by changing the temperature of the medium.

[0105] Therefore, an oligon...

Embodiment 3

[0135] The Gram-positive bacterium Bacillus subtilis is widely used as an industrial bioreactor for the production of a range of heterologous proteins. It was therefore of interest to test whether the random spacer method of the present invention could also be used to generate a promoter library for this organism. The consensus sequence of Bacillus subtilis is very similar to the consensus sequence of E. coli and Lactococcus lactis, so we can test whether this method is effective for Bacillus subtilis by subcloning a large number of CP promoters into the promoter cloning vector of Bacillus subtilis also works, it can then be investigated whether 1) the CP promoter is active in Bacillus subtilis, and 2) whether the spacer between the consensus sequences also plays an important role in the promoter strength in this organism. We chose to use the promoter cloning vector pDG268, which was designed to integrate the promoter fusion with lacZ into the amy locus on the Bacillus subtili...

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Abstract

An artificial promoter library for a selected organism or group of organisms is constructed as a mixture of double stranded DNA fragments, the sense strands of which comprise at least two consensus sequences of efficient promoters from said organism or group of organisms, or parts thereof comprising at least half of each, and surrounding or intermediate nucleotide sequences (spacers) of variable length in which at least 7 nucleotides are selected randomly among the nucleobases A, T, C and G. The sense strands of the double stranded DNA fragments may also include a regulatory DNA sequence imparting a specific regulatory feature, such as activation by a change in the growth conditions, to the promoters of the library. Further, they may have a sequence comprising one or more recognition sites for restriction endonucleases added to one of or both their ends. The selected organism or group or organisms may be selected from prokaryotes and from eukaryotes; and in prokaryotes the consensus sequences to be retained most often will comprise the -35 signal (-35 to -30): TTGACA and the -10 signal (-12 to -7): TATAAT or parts of both comprising at least 3 conserved nucleotides of each, while in eukaryotes said consensus sequences should comprise a TATA box and at least one upstream activation sequence (UAS). Such artificial promoter libraries can be used i.a. for optimizing the expression of specific genes in various selected organisms.

Description

field of invention [0001] The present invention relates to artificial promoter libraries and methods for constructing artificial promoter libraries useful in a selected organism or group of organisms. The present invention also relates to each novel promoter obtained from such a library. In addition, the present invention relates to a method for optimizing the expression of a gene in a selected organism by using a promoter obtained from such an artificial promoter library in that organism. In principle, the artificial promoter libraries of the present invention can be constructed for use in any living organism, but at present they are mostly used to regulate gene expression in microorganisms. For the purposes of the present invention, the term "microorganism" shall broadly include prokaryotic organisms such as bacteria, and eukaryotic microorganisms such as yeast, other fungi and cell lines of higher organisms. Background of the invention [0002] Although genetic engineer...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/11C12N15/63C12N15/09C12N15/10
CPCC12N15/63C12N15/1051
Inventor 彼得·鲁戴尔·简森卡林·哈默
Owner 彼得·鲁戴尔·简森