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HTP genomic engineering platform for improving escherichia coli

A technology of Escherichia coli and genetic engineering, which is applied in the field of high-throughput microbial genetic engineering and can solve the problem that researchers do not have genetic engineering tools.

Pending Publication Date: 2020-03-31
ZYMERGEN INC
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clearly, traditional strain improvement methods have reached a plateau for this biological system, but researchers do not have the genetic engineering tools needed to cross this plateau

Method used

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  • HTP genomic engineering platform for improving escherichia coli
  • HTP genomic engineering platform for improving escherichia coli
  • HTP genomic engineering platform for improving escherichia coli

Examples

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example

[0898] The following examples are provided to illustrate various embodiments of the present disclosure and are not intended to limit the present disclosure in any way. Those skilled in the art will recognize that changes therein and other uses are encompassed within the spirit of the disclosure as defined by the scope of the claims.

[0899] Specifically, Examples 1-9 are HTP genetic engineering platforms in coryneform bacteria. However, a similar procedure has been tailored for E. coli and was successfully performed by the present inventors.

[0900] A brief table of contents is provided below to assist the reader only. This list is not intended to limit the scope of the examples or disclosure of this application.

[0901] Table 5.1 - Table of Contents for Example Sections

[0902]

[0903]

example 1

[0904] Example 1: Demonstration of HTP transformation and SNP library creation of coryneform bacteria

[0905] This example illustrates an embodiment of the HTP genetic engineering method of the present disclosure. Host cells are transformed with multiple SNP sequences of different sizes, all targeting different regions of the genome. The results demonstrate that the methods of the present disclosure are capable of producing rapid genetic changes of any kind throughout the entire genome of a host cell.

[0906] A. Cloning of Transformation Vectors

[0907] A variety of SNPs were randomly selected from Corynebacterium glutamicum (ATCC21300) and cloned into a Corynebacterium cloning vector using yeast homologous recombination cloning technology to assemble the vector, wherein each SNP is flanked by a direct repeat region, as described above in "Assembly / Cloning of Custom Plasmids" section and as described in image 3 described in.

[0908] The SNP cassette used in this exam...

example 2

[0929] Example 2: HTP Genome Engineering - Implementation of SNP Libraries to Repair / Improve Industrial Microbial Strains

[0930] This example illustrates several aspects of the SNP exchange library in the HTP strain improvement program of the present disclosure. In particular, the examples illustrate several conceived approaches to rehabilitate currently existing industrial strains. This example describes up-swing and down-swing approaches to explore the phenotypic solution space arising from multiple genetic differences that may exist between "basic", "intermediate" and industrial strains.

[0931] A. Identification of SNPs in Diversity Pools

[0932] An exemplary strain improvement program using the methods of the present disclosure was performed on an industrial microbial strain (referred to herein as "C"). The diversity pool strains used in this procedure are indicated by A, B and C. Strain A represents the original production host strain prior to any mutagenesis. St...

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Abstract

The present disclosure provides a HTP genomic engineering platform for improving escherichia coli. that is computationally driven and integrates molecular biology, automation, and advanced machine learning protocols. The integrative platform utilizes a suite of HTP molecular tool sets to create HTP genetic design libraries, which are derived from, inter alia, scientific insight and iterative pattern recognition.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Application No. 62 / 515,870, filed June 6, 2017, which is hereby incorporated by reference in its entirety for all purposes. technical field [0003] The invention relates to high-throughput (HTP) microbial genetic engineering transformation for Escherichia coli. The disclosed HTP genetic engineering platform is computer driven and integrates molecular biology, automation and advanced machine learning solutions. This integrated platform utilizes a set of HTP molecular tools to create HTP genetic design libraries derived using inter alia scientific insights and iterative pattern recognition. [0004] Statement Regarding Sequence Listing [0005] The Sequence Listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence lis...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12N15/73
CPCC12N15/1058C12N15/1079C12N15/73
Inventor Z·塞尔贝尔E·J·迪安S·曼彻斯特K·戈拉E·谢尔曼A·金博尔S·希捷卡B·弗勒旺T·特赖诺尔M·弗拉斯曼R·豪斯哈尔特S-A·摩根M·布莱斯P·拉马克里希南K·罗特席尔德-曼奇内里Y·基姆M·戴维斯C·威斯纳维斯基P·韦斯特福尔
Owner ZYMERGEN INC
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