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Modular genomes for synthetic biology and metabolic engineering

a technology of metabolic engineering and modules, applied in the field of methods and compositions for genetically modified microorganisms, can solve the problems of increasing the difficulty of manipulating multiple genes and/or regulatory elements, lack of order, etc., and achieves the effect of maximizing or minimizing protein production, facilitating replacement, deletion and/or addition

Inactive Publication Date: 2007-10-18
BRITISH COLUMBIA CANCER AGENCY BRANCH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Such lack of order increases the difficulties of manipulating multiple genes and / or regulatory elements that may be involved in the same metabolic pathway.

Method used

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  • Modular genomes for synthetic biology and metabolic engineering
  • Modular genomes for synthetic biology and metabolic engineering
  • Modular genomes for synthetic biology and metabolic engineering

Examples

Experimental program
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Effect test

example i

Use of H. influenzae as Genome Donor to an E. coli Host

[0051] In this example, H. influenzae was selected as a donor organism because of its free living status and its relatively compact genome (1.83 Mbp). The strain of H. influenzae employed is Rd KW20, which is kanamycin resistant and RecA negative (to eliminate the possibility of confounding homologous recombination). E. coli was selected as the host organism, specifically the HMS174 strain (Novagen), as it has K12 background, supports IPTG inducible recombinant protein expression, and is RecA negative. H. influenzae and E. coli are closely related, commensal gammaproteobacteria and the complete genome sequence is available for both organisms [(Science 269, 496-512 (1995); Science 277, 1453-1452 (1997)].

[0052] A BAC library was constructed from an MboI partial digest of H. influenzae gDNA. BAC clones from this library were end sequenced at high redundancy (>200× clone coverage) and mapped to the reference H. influenzea genome s...

example ii

In Vivo Assembly of Episomal Elements in E. coli

[0054] In this example episomal elements that contain mutant LoxP sites are constructed using standard molecular biology procedures. These constructs are transformed sequentially into an E. coli host and fusion is mediated by induction of Cre expression within the host cell. Separately, Bacterial Artificial Chromosomes (BACs) containing large segments of the H. influenzae genome are retrofit with mutant lox sites and selectable markers using the RED / ET system. BACs retrofitted in this manner are suitable for serial recombination in Cre expressing RED / ET E. coli host cells.

[0055] A short DNA segment with an EcoRI compatible overhang on one end plus a HindIII compatible overhang on the opposite end, and containing a LoxP site that has both an LE arm mutant (ATAAC to TACCG) and a spacer mutant (C to G at spacer position 2 and A to C at spacer position 7) was ligated into EcoRI / HindIII cut and gel purified pET19b expression vector. This ...

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Abstract

The invention provides methods and compositions for assembling a modular replacement genome in a host microorganism. After such assembly, the host organism's genome is inactivated or ablated to permit full control of host cellular functions by the replacement genome. A modular replacement genome comprises an assembly of nucleic acid fragments, or segments, derived from one or more natural organisms or from synthetic polynucleotides or from a combination of both. Such an assembly, or set, of segments making up a replacement genome comprises a substantially complete set of genes and regulatory elements for carrying out minimal life functions under predefined culture conditions. The invention provides modular genomes having modules that are amenable to facile replacement, deletion, and / or additions. Such modules may be synthetic polynucleotides and may be designed for controlling gene content, excluding of genes that encode inhibitors or otherwise undesirable competing enzymes that divert a host cell from desired metabolic / synthetic processes; modifying codon usage to maximize or minimize protein production; modifying regulatory elements, including promoters, enhancers, repressors, activator, or the like, to modulate gene expression; balancing enzymatic and transport activities to optimize fluxes of substrates, intermediates, and products in metabolic pathways, and like objectives.

Description

[0001] This application claims priority from U.S. provisional applications Ser. Nos. 60 / 725,630 filed 13 Oct. 2005, which is hereby incorporated by reference in its entirety.FIELD OF THE INVENTION [0002] The invention relates generally to methods and compositions for genetically modifying microorganisms, and more particularly, to methods and compositions for selectively modifying genomic regions containing predefined sets of genes and / or genetic elements. BACKGROUND [0003] Metagenomics and the development of techniques for the facile production and manipulation of large fragments of DNA have spurred interest in engineering microorganisms for a host of important industrial and medical applications, e.g. Lorenz et al, Nature Reviews Microbiology, 3: 510-516 (2005); Branda et al, Developmental Cell, 6: 7-28 (2004); Kodumal et al, Proc. Natl. Acad. Sci., 101: 15573-15578 (2004); Tian et al, Nature, 432: 1050-1054 (2004). Metagenomics is the application of genomics techniques to the stud...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N1/21C07H21/04C12N15/00C12N15/74
CPCC12N15/70C12N2800/30C12N15/90
Inventor HOLT, ROBERT A.
Owner BRITISH COLUMBIA CANCER AGENCY BRANCH
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