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Method for the positive selection of chromosomal mutations in C1 metabolizing bacteria via homologous recombination

a chromosomal mutation and metabolizing bacteria technology, applied in the field of molecular biology, can solve the problems of low product yield, low economic success of scp, and slight commercial success of epoxidation of alkenes

Inactive Publication Date: 2006-03-16
EI DU PONT DE NEMOURS & CO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ts. Only epoxidation of alkenes has experienced slight commercial success due to low product yields, toxicity of products, and the large amount of cell mass required to generate produc
33). However, SCP has not been economically successful thus far due to the relatively high cost of producing microbial protein, as compared to agriculturally derived protein (i.e., soy prot
ein). This makes SCP a relatively low value product whose economic production cannot tolerate heavy bioprocessing
rocess. Microbial biomass produced by methanotrophic bacteria is typically very high in protein content (˜70-80% by weight), which can restrict the direct use of this protein to certain types of anim
16a, however, is currently limited by the lack of efficient tools for generating defined mutants by homologous recombination.
In theory, this type of recombination event is easily detected on a selective medium; however, performing allelic exchange in C1 metabolizing microorganisms has been relatively cumbersome due to the organisms' slow growth rates and the rarity of double-crossover events (which require extensive screening to isolate an allelic-exchange mutant).
None of these previous studies, however, have utilized positive selection as a method for the rapid identification of allelic exchange mutants.
nas. However, no one has previously attempted to use sacB in a positive selection method for C1 metabolizing bact

Method used

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  • Method for the positive selection of chromosomal mutations in C1 metabolizing bacteria via homologous recombination
  • Method for the positive selection of chromosomal mutations in C1 metabolizing bacteria via homologous recombination
  • Method for the positive selection of chromosomal mutations in C1 metabolizing bacteria via homologous recombination

Examples

Experimental program
Comparison scheme
Effect test

example 1

Growth of Methylomonas 16a

[0224] Example 1 summarizes the standard conditions used for growth of Methylomonas sp. 16a (ATCC# PTA-2402), as described in U.S. Pat. No. 6,689,601.

Methylomonas Strain and Culture Media

[0225] The growth conditions described below were used throughout the experimental Examples for treatment of Methylomonas 16a, unless conditions were specifically described otherwise.

[0226]Methylomonas 16a is typically grown in serum stoppered Wheaton bottles (Wheaton Scientific, Wheaton, Ill.) using a gas / liquid ratio of at least 8:1 (i.e., 20 mL of Nitrate liquid “BTZ” media in 160 mL total volume). The standard gas phase for cultivation contained 25% methane in air, although methane concentrations can vary ranging from about 5-50% by volume of the culture headspace. These conditions comprise growth conditions and the cells are referred to as growing cells. In all cases, the cultures were grown at 30° C. with constant shaking in a Lab-Line rotary shaker unless otherwi...

example 2

Construction of a Positive-Selective Suicide Vector for Methylomonas sp. 16a

[0230] The construction of chromosomal mutations within the Methylomonas genome required the use of suicide vectors. Thus, a modified version of the conditional replication vector pGP704 was created, comprising a npr-sacB cassette.

pGP704 as a Vector Backbone for the C1 Chromosomal Integration Vector

[0231] The plasmid pGP704 (Miller and Mekalanos, J. Bacteriol., (170): 2575-2583 (1988); FIG. 4) was chosen as a suitable vector backbone for the C1 chromosomal integration vector, since it could be used as a vehicle to transfer replacement nucleotide sequences of interest into Methylomonas sp. 16a via conjugation. Plasmid pGP704 is a derivative of pBR322 that is AmPR but has a deletion of the pBR322 origin of replication (oriE1). Instead, the plasmid contains a cloned fragment containing the origin of replication of plasmid R6K. The R6K origin of replication (oriR6K) requires the Π protein, encoded by the pir...

example 3

Construction of pGP704::sacB::crtN2::EZ::TN™

[0244] The native Methylomonas gene encoding crtN2 was disrupted by EZ::TN™ (Epicenter Technologies, Madison, Wis.) to generate an appropriate replacement nucleotide sequence of interest (i.e., re-NSI) in the chromosomal integration vector pGP704::sacB. This construct, pGP704::sacB::crtN2::EZ::TN™ was then utilized in Examples 4 and 5 to test the homologous recombination abilities of Methylomonas sp. 16a, using the one-step selection strategy to identify allelic exchange mutants.

PCR Amplification and Cloning of the crtN2 DNA Fragment into pGP704::sacB

[0245] DNA primers crtN#2-ctg 288+1 kB / Bg / ll and crtN#2-ctg 288+1kB / Xbal were used to amplify a ˜3.1 kB DNA fragment from Methylomonas sp. 16a chromosomal DNA, comprising SEQ ID NO:3 flanked by approximately 1 Kb of DNA.

crtN#2-ctg 288 + 1kB / Bg / II:5′-AGATCTTTCCGGTCATGCTGGAGTTGGG-3′(SEQ ID NO:6)crtN#2-ctg 288 + 1kB / XbaI:5′-TCTAGATTGCAGCTCAAGCGATTCGG-3′(SEQ ID NO:7)

[0246] As described in Exa...

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Abstract

A method for the positive selection of allelic exchange mutants is provided for C1 metabolizing bacteria. The chromosomal integration vectors, based on the pGP704 suicide vector, comprise at least one genetic selectable marker and the sacB gene, encoding levansucrase. A one- and two-step selection strategy is provided for the facile identification of double-crossover mutations in C1 metabolizing bacteria. This methodology enables production of “markerless” transformants, such that multiple rounds of mutation can be performed. Optimized conditions for conjugal transfer, homologous recombination, transformant purification, and screening are also presented.

Description

[0001] This application claims the benefit of U.S. Provisional Application No. 60 / 527,877 filed Dec. 8, 2003 and U.S. Provisional Application No. 60 / 527,083 filed Dec. 3, 2003.FIELD OF THE INVENTION [0002] The present invention relates to the field of molecular biology and the use of selection vectors for the identification of recombinant bacteria containing heterologous DNA. More specifically, vectors have been constructed for the positive selection of double-crossover mutants in C1 metabolizing bacteria. BACKGROUND INFORMATION [0003] There are a number of microorganisms that utilize single carbon substrates as their sole energy source. Such microorganisms are referred to herein as “C1 metabolizers”. These organisms are characterized by the ability to use carbon substrates lacking carbon to carbon bonds as a sole source of energy and biomass. All C1 metabolizing microorganisms are generally classified as methylotrophs. Methylotrophs may be defined as any organism capable of oxidizi...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/74C12N9/00C12N9/02C12N9/10C12N9/90C12N15/52C12P23/00
CPCC12N9/00C12N9/0004C12N9/001C12N9/0071C12N9/0083C12N9/1051C12Y205/01029C12N9/90C12N15/52C12N15/74C12P23/00C12R1/26C12N9/1085C12R2001/26C12N1/205
Inventor SHARPE, PAMELA
Owner EI DU PONT DE NEMOURS & CO
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