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Regulatable gene expression

A gene expression and adjustable technology, which is applied in the determination/testing of microorganisms, growth factors/inducing factors, nucleotide libraries, etc., and can solve the problem that large groups are not effective

Active Publication Date: 2016-03-09
BIOSYNTIA APS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This system is useful for smaller cell populations (approximately 10 5 cells) but less so for larger populations, with cells containing about 10 6 10-30% false positives observed for libraries of cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Example 1. Construction of double riboswitch selection vector pGEBO5.1

[0155] pGEBO5.1 is a third generation selection vector based on the second generation selection vector pGEBO4.1, and pGEBO4.1 is based on the first generation selection vector pGEBO4. pGEBO4 comprises the vector pGEBO2 containing the 'riboswitch platform'.

[0156] Construction of pGEBO2: 221 base pair DNA sequence constituting the 'riboswitch platform' ( figure 1 ) was artificially synthesized by Integrated DNA Technologies and delivered on the pIDTSMART vector, resulting in pGEBO2. In addition to the riboswitch platform, the 5' end of the riboswitch includes a Flippase Recognition Target (FRT) sequence (5'-GAAGTTCCTATTTCTCTAGAAAGTATAGGAACTTC-3' (SEQ ID NO: 19)).

[0157] Construction of pGEBO4: pGEBO4 was established by circular USER fusion of three PCR products: (1) FRT-riboswitch platform amplified from pGEB02 with primers GEBO4 / 5 (forward / reverse); (2) with primer GEBO6 / 2 region containing...

Embodiment 2

[0160] Example 2. Determination of spontaneous resistance in single and double selection systems

[0161] pGEBO5.1 was transformed into E. coli Dh10B cells (forming EcpGEBO5.1 ) using standard methods known to the skilled person. To test whether the dual selection system resolved the problem of spontaneous resistance, up to 1.3x10 8 Plating of EcpGEBO5.1 cells ('double selection'). As a control, plating on plates containing only chloramphenicol (30 μg / mL) or spectinomycin (80 μg / mL) was each performed in triplicate (‘single selection’) (see Table 1). Each plate was incubated for 72 hours. In the 10 plates subjected to double selection, no colonies were observed within the first 48 hours of incubation. During the 24 h incubation, each of the three plates containing only chloramphenicol produced numerous colonies, as did the plate containing only spectinomycin. When the 1.3×10 8Spontaneous resistance was observed in the double selection system when each cell was plated and ...

Embodiment 3

[0164] Example 3. Growth response of EcpGEBO5.1 to different xanthine alkaloids

[0165] The growth response of EcpGEBO5.1 to 4 different xanthine alkaloids was determined empirically using a selection assay with different concentrations of the xanthine-based compounds theophylline, xanthine, theobromine and caffeine.

[0166] Procedure: Plate 300-400 colony-forming units in a mixture containing ampicillin (50 µg / mL), spectinomycin (80 µg / mL), chloramphenicol (30 µg / mL), and xanthine-based compounds at indicated concentrations (see Table 2) On the flat plate: predetermine the dissociation constant (K d ) (Jenison et al., 1994). No selection refers to plates without spectinomycin and chloramphenicol. (+) means growth was observed. (-) means that no growth was observed. (approximately) means that reduced growth was observed.

[0167] Table 2 - Characterization of the specificity of the riboswitch aptamer domain in pGEBO5.1.

[0168]

[0169] These results indicate that ...

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Abstract

The present invention relates to a regulatable gene expression construct comprising a nucleic acid molecule comprising two or more regulation sequences encoding respective RNA molecules comprising a riboswitch responsive to an effector compound, said riboswitch being operably linked to a respective coding region which encodes a respective modulator compound for modulating the action of a respective growth regulator compound and each said riboswitch in each regulation sequence being selected be responsive to the same effector compound to trigger expression of its respective modulator compound. The invention also relates to a method of using the regulatable gene expression construct for selecting from a metagenomic library a primary modulator compound which effects a chemical transformation of a substrate into said effector compound or transports said effector compound into a micro-organism comprising the regulatable gene expression construct.

Description

technical field [0001] The present invention relates to a regulatable gene expression construct and methods of using said regulatable gene expression construct in in vivo selection procedures. Background technique [0002] Biocatalysts are biologically produced catalysts that activate or accelerate the rate of chemical reactions. Biocatalysis is becoming an increasingly important tool in the chemical industry, especially in the paper, leather, personal care and detergent industries (Sanchez and Demain, 2011). Biocatalysts can comprise whole-cell systems as well as isolated enzymes; the latter are generally classified as industrial enzymes and biotransformation enzymes. [0003] Biocatalytic enzymes play an even more important role in organic synthetic methods. An increasing number of industrial products (from pharmaceutical intermediates to bulk chemicals) are manufactured by processes involving one or more biocatalytic steps, thereby predicting the emergence of a signific...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/10C12N15/63C12N15/67C40B40/02C40B40/06
CPCC12N15/1093C12N15/63C12N15/67C07K14/475C12N15/1034C12N15/1055C12N15/1079C12N15/635
Inventor 汉斯·贾斯帕·吉尼莫滕·萨默
Owner BIOSYNTIA APS
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