Method for creating a genomic library enriched for bacillus and identification of novel cry toxins

Abstract

The disclosure is directed to an approach for discovering novel insecticidal proteins from highly heterogeneous environmental sources. The methodology utilizes metagenomic enrichment procedures that increase the representation of specific genera of bacteria, prior to deep sequencing, in order to access novel variants of insecticidal proteins. In aspects, the taught methods are useful for creating large genomic libraries enriched for Bacillus sp. The enriched genomic libraries are useful for the discovery of novel cry toxin genes, which enable the identification of novel Cry proteins.

What is claimed is: 
     
         1 . A population of germinating microbial cells in bulk liquid culture, wherein the population comprises at least 90% thermotolerant microorganisms. 
     
     
         2 . The population of  claim 1 , wherein the thermotolerant microorganisms are of the order Bacillales. 
     
     
         3 . The population of  claim 1 , wherein the thermotolerant microorganisms are of the genus  Bacillus.    
     
     
         4 . The population of  claim 1 , wherein the population comprises at least 80% thermotolerant  Bacillus  sp. 
     
     
         5 . The population of  claim 1 , wherein the population comprises at least 90% thermotolerant  Bacillus  sp. 
     
     
         6 . The population of  claim 1 , wherein the thermotolerant microorganisms are obtained from soil. 
     
     
         7 . The population of  claim 1 , wherein the thermotolerant microorganisms are obtained from arid soil. 
     
     
         8 . The population of  claim 1 , wherein the thermotolerant microorganisms are obtained from a soil sample, and wherein the population comprises at least 100-fold more thermotolerant microorganisms as an overall percentage of microorganisms, compared to a control soil sample. 
     
     
         9 . The population of  claim 1 , wherein the population is comprised in a laboratory medium. 
     
     
         10 . The population of  claim 1 , wherein the population is contained in a flask or tube. 
     
     
         11 . The population of  claim 1 , wherein the population is contained in a conical centrifuge tube. 
     
     
         12 . The population of  claim 1 , wherein the microbial cells are present at a non-natural concentration. 
     
     
         13 . A composition comprising an isolated genomic DNA mixture, wherein at least 90% of the total genomic DNA is from thermotolerant microorganisms, and wherein the composition is contained in discrete microliter volumes on a solid substrate. 
     
     
         14 . The composition of  claim 13 , wherein the thermotolerant microorganisms are of the order Bacillales. 
     
     
         15 . The composition of  claim 13 , wherein the thermotolerant microorganisms are of the genus  Bacillus.    
     
     
         16 . The composition of  claim 13 , wherein at least 80% of the total genomic DNA is from thermotolerant  Bacillus  sp. 
     
     
         17 . The composition of  claim 13 , wherein at least 90% of the total genomic DNA is from thermotolerant  Bacillus  sp. 
     
     
         18 . The composition of  claim 13 , wherein the thermotolerant microorganisms are obtained from soil. 
     
     
         19 . The composition of  claim 13 , wherein the thermotolerant microorganisms are obtained from arid soil. 
     
     
         20 . The composition of  claim 13 , wherein the thermotolerant microorganisms are obtained from a soil sample, and wherein the composition comprises at least 100-fold more genomic DNA from thermotolerant microorganisms as an overall percentage, compared to genomic DNA isolated from a control soil sample. 
     
     
         21 . The composition of  claim 13 , wherein the solid substrate is a 96 well plate, a flask, or a tube. 
     
     
         22 . A composition comprising:
 (i) an isolated genomic DNA mixture, wherein at least 90% of the total genomic DNA is from thermotolerant microorganisms; and   (ii) a PCR reagent.   
     
     
         23 . The composition of  claim 22 , wherein the thermotolerant microorganisms are of the order Bacillales. 
     
     
         24 . The composition of  claim 22 , wherein the thermotolerant microorganisms are of the genus  Bacillus.    
     
     
         25 . The composition of  claim 22 , wherein at least 80% of the total genomic DNA is from thermotolerant  Bacillus  sp. 
     
     
         26 . The composition of  claim 22 , wherein at least 90% of the total genomic DNA is from thermotolerant  Bacillus  sp. 
     
     
         27 . The composition of  claim 22 , wherein the thermotolerant microorganisms are obtained from soil. 
     
     
         28 . The composition of  claim 22 , wherein the thermotolerant microorganisms are obtained from arid soil. 
     
     
         29 . The composition of  claim 22 , wherein the thermotolerant microorganisms are obtained from a soil sample, and wherein the composition comprises at least 100-fold more genomic DNA from thermotolerant microorganisms as an overall percentage, compared to genomic DNA isolated from a control soil sample. 
     
     
         30 . The composition of  claim 22 , wherein the composition is contained in a 96 well plate, flask, or tube. 
     
     
         31 . A composition comprising an isolated megaplasmid DNA mixture, wherein at least 90% of the megaplasmid DNA is from thermotolerant microorganisms, and wherein the composition is contained in discrete microliter volumes on a solid substrate. 
     
     
         32 . The composition of  claim 31 , wherein at least 80% of the overall DNA in the composition is megaplasmid DNA. 
     
     
         33 . The composition of  claim 31 , wherein at least 90% of the overall DNA in the composition is megaplasmid DNA. 
     
     
         34 . The composition of  claim 31 , wherein the thermotolerant microorganisms are of the order Bacillales. 
     
     
         35 . The composition of  claim 31 , wherein the thermotolerant microorganisms are of the genus  Bacillus.    
     
     
         36 . The composition of  claim 31 , wherein at least 80% of the total megaplasmid DNA is from thermotolerant  Bacillus  sp. 
     
     
         37 . The composition of  claim 31 , wherein at least 90% of the total megaplasmid DNA is from thermotolerant  Bacillus  sp. 
     
     
         38 . The composition of  claim 31 , wherein the composition comprises less than 10% linear double-stranded DNA as a percentage of overall DNA. 
     
     
         39 . The composition of  claim 31 , wherein the thermotolerant microorganisms are obtained from soil. 
     
     
         40 . The composition of  claim 31 , wherein the thermotolerant microorganisms are obtained from arid soil. 
     
     
         41 . The composition of  claim 31 , wherein the thermotolerant microorganisms are obtained from a soil sample, and wherein the composition comprises at least 100-fold more megaplasmid DNA from thermotolerant microorganisms as an overall percentage, compared to megaplasmid DNA isolated from a control soil sample. 
     
     
         42 . The composition of  claim 31 , wherein the solid substrate is a 96 well plate, flask, or tube. 
     
     
         43 . A composition comprising:
 (i) an isolated megaplasmid DNA mixture, wherein at least 90% of the total megaplasmid DNA is from thermotolerant microorganisms; and   (ii) a PCR reagent.   
     
     
         44 . The composition of  claim 43 , wherein at least 80% of the overall DNA in the composition is megaplasmid DNA. 
     
     
         45 . The composition of  claim 43 , wherein at least 90% of the overall DNA in the composition is megaplasmid DNA. 
     
     
         46 . The composition of  claim 43 , wherein the thermotolerant microorganisms are of the order Bacillales. 
     
     
         47 . The composition of  claim 43 , wherein the thermotolerant microorganisms are of the genus  Bacillus.    
     
     
         48 . The composition of  claim 43 , wherein at least 80% of the total megaplasmid DNA is from thermotolerant  Bacillus  sp. 
     
     
         49 . The composition of  claim 43 , wherein at least 90% of the total megaplasmid DNA is from thermotolerant  Bacillus  sp. 
     
     
         50 . The composition of  claim 43 , wherein the composition comprises less than 10% linear double-stranded DNA as a percentage of overall DNA. 
     
     
         51 . The composition of  claim 43 , wherein the thermotolerant microorganisms are obtained from soil. 
     
     
         52 . The composition of  claim 43 , wherein the thermotolerant microorganisms are obtained from arid soil. 
     
     
         53 . The composition of  claim 43 , wherein the thermotolerant microorganisms are obtained from a soil sample, and wherein the composition comprises at least 100-fold more megaplasmid DNA from thermotolerant microorganisms as an overall percentage, compared to megaplasmid DNA isolated from a control soil sample. 
     
     
         54 . The composition of  claim 43 , wherein the composition is contained in a 96 well plate, flask, or tube.