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Methods of obtaining genetic competence in bacillus cells

A technology of Bacillus genus and competence, which is applied in the field of obtaining genetic competence, and can solve problems such as the inability to prove the transformation of Bacillus licheniformis

Inactive Publication Date: 2016-09-21
NOVOZYMES AS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Furthermore, these investigators were unable to demonstrate transformation by natural competence in Bacillus licheniformis ATCC 10716 (Gwinn and Thorne, 1964, supra)

Method used

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  • Methods of obtaining genetic competence in bacillus cells
  • Methods of obtaining genetic competence in bacillus cells
  • Methods of obtaining genetic competence in bacillus cells

Examples

Experimental program
Comparison scheme
Effect test

specific Embodiment approach

[0260] DNA sequencing

[0261] Using Applied Biosystems Model 3130X Genetic Analyzer (3130X Genetic Analyzer) (Applied Biosystems, Foster City, CA, USA), utilize dye terminator chemistry (dye terminatorchemistry) (Giesecke etc., 1992, Journal of Virol.Methods 38:47- 60) Perform DNA sequencing. Sequences were assembled with sequencing specific primers using phred / phrap / consed (University of Washington, Seattle, WA, USA).

[0262] coli strain

[0263] Use ONE TOP10 chemically competent Escherichia coli cells (Invitrogen Corporation, Carlsbad, CA, USA), Competent E. coli cells (Stratagene, La Jolla, CA, USA), XL1-Blue competent E. coli cells (Stratagene, La Jolla, CA, USA), and Gold supercompetent E. coli cells (Stratagene, La Jolla, CA, USA) were used for routine plasmid construction and propagation.

[0264] Bacillus strains

[0265] B. subtilis 168Δ4 is ​​derived from B. subtilis type strain 168 (BGSC 1A1, Bacillus Genetic Stock Center, Columbus, OH, USA) and has dele...

Embodiment 1

[0283] Example 1: Determination of the Genome Sequence of Bacillus licheniformis Strain SJ1904

[0284]From contigs generated using 454 DNA sequencing technology (Margulies et al., 2005, Nature 437:376-380), random paired reads using Sanger sequencing technology, and for closing gaps and resolving repetitive sequences Reads from PCR fragments of genomic DNA, determining the genome sequence of the complete chromosome of Bacillus licheniformis strain SJ1904. Sequencing data was assembled using Phrap and edited and viewed in Consed. Gene models were predicted from genomic DNA sequences using Glimmer (Delcher et al., 1999, Nucleic Acids Research 27:4636-4641). Use an E-value threshold of 1X10 -5 BLASTP for machine annotation of gene models by comparison with the non-redundant database PIR-NREF (Wu et al., 2002, Nucleic Acids Research 30:35-37).

Embodiment 2

[0285] Example 2: Identification of Bacillus licheniformis M.Bli1904II DNA methyltransferase gene

[0286] The deduced amino acid sequence of the gene model of B. licheniformis strain SJ1904 was compared with the protein sequence from REBASE (Roberts, R.J., Macelis, M., Rebase. 2005) using BLASTP (Altschul et al., 1997, Nucleic Acids Research 25:3389-3402). Compare. Because DNA methyltransferases have a moderate level of sequence conservation, this analysis identified all putative DNA methyltransferases in this genome. The cytosine-specific DNA methyltransferase signature in M.Bli1904II was identified using Prints-S version 16 performed by InterProScan version v3.3. In addition, six highly conserved motifs (motifs) found in cytosine-specific DNA methyltransferases were also conserved in Bacillus licheniformis M. Bli1904II DNA methyltransferase.

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Abstract

The present invention methods of obtaining genetic competence in bacillus cells and in particular relates to methods of obtaining genetic competence in non-competent Bacillus cells for their transformation with exogenous DNA.

Description

[0001] The application of the present invention is based on the invention patent with the application date of December 19, 2007, the application number 200780051674.2 (the international application number is PCT / US2007 / 088186), and the name is "Methods for Obtaining Genetic Competence in Bacillus Cells" Divisional application of the application. [0002] References to Sequence Listings [0003] This application contains a Sequence Listing in computer readable format. The computer readable format is incorporated herein by reference. technical field [0004] The present invention relates to methods for obtaining genetic competence in non-competent Bacillus cells. Background technique [0005] Genetic competence is the physiological state in which exogenous DNA can be internalized, resulting in transformation events (Berka et al., 2002, Mol. Microbiol. 43:1331-45), but with 2 Manual transformations are handled differently. Natural competence has been observed in both Gram-p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21
CPCC07K14/32
Inventor 兰迪·伯卡米歇尔·马兰塔玛丽亚·唐巴巴拉·彻里
Owner NOVOZYMES AS
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