Evolution of bt toxins

a technology of toxins and toxins, applied in the field of bt toxins, can solve problems such as the problem of pest resistance populations becoming a problematic phenomenon

Active Publication Date: 2018-03-01
PRESIDENT & FELLOWS OF HARVARD COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some target pests have developed resistance to this and to related Bt toxins, and resistant populations of pests have become a problematic phenomenon.

Method used

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  • Evolution of bt toxins
  • Evolution of bt toxins
  • Evolution of bt toxins

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Materials and Methods

[0125]General methods. All PCR reactions were performed using PfuTurbo Cx Hotstart DNA polymerase (Agilent Technologies), VeraSeq ULtra DNA polymerase (Enzymatics) or Phusion U Hot Start DNA Polymerase (Life Technologies). Water was purified using a MilliQ water purification system (Millipore, Billerica MA). All plasmids and selection phages were constructed using USER cloning (New England Biolabs). Genes were synthesized as bacterial codon-optimized gBlocks Gene Fragments (Integrated DNA Technologies). All DNA cloning was carried out using NEB Turbo cells (New England Biolabs).

[0126]Electrocompetent strain preparation. Strain S1030 was used for all luciferase and plaque assays, as well as continuous evolution experiments. The glycerol stock of S1030 cells was used to seed a 2 mL overnight culture using 2×YT media supplemented with 10 μg / mL tetracycline (Sigma Aldrich), 50 μg / mL streptomycin (Sigma Aldrich), 10 μg / mL fluconazole (TCI America), and 10 μg / mL ampho...

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Abstract

The disclosure provides amino acid sequence variants of Bacillus thuringiensis (Bt) toxins and methods of producing the same. Some aspects of this disclosure provide methods for generating Bt toxin variants by continuous directed evolution. Some aspects of this disclosure provide compositions and methods for pest control using the disclosed variant Bt toxins.

Description

RELATED APPLICATIONS[0001]This application claims priority to U.S. provisional applications, U.S. Ser. No. 62 / 196,253, filed Jul. 23, 2015, entitled “EVOLUTION OF BT TOXINS,” and U.S. Ser. No. 62 / 305,497, filed Mar. 8, 2016, entitled “EVOLUTION OF BT TOXINS,” each application which is incorporated herein by reference.GOVERNMENT SUPPORT[0002]This invention was made with government support under grant numbers HR0011-11-2-0003 and N66001-12-C-4207 awarded by the Defense Advanced Research Projects Agency (DARPA). The government has certain rights in the invention.BACKGROUND OF THE INVENTION[0003]Bacillus thuringiensis (Bt) is a gram-positive bacterium that produces crystalline inclusion bodies (Bt toxins) during sporulation, which exhibit potent insecticidal activity through disruption of the osmotic balance in the insect midget as a consequence of membrane insertion and pore formation (FIG. 1). A limited number of Bt toxins have become widespread biological alternatives to chemical ins...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/325C12N15/82A01N63/02C12N15/10A01N63/50
CPCC07K14/325C12N15/1058A01N63/02C12N15/8286A01N63/50Y02A40/146A01N63/23
Inventor LIU, DAVID R.BADRAN, AHMED HUSSEINGUZOV, VICTORMALVAR, TOMVISHWANATH, PRASHANTHNAGEOTTE, JEFFHUAI, QINGKEMP, MELISSA
Owner PRESIDENT & FELLOWS OF HARVARD COLLEGE
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