Targeting sequence for paenibacillus-based endospore display platform

A Bacillus genus, sequence technology, applied to methods using spores, methods based on microorganisms, immobilized on or in biological cells, etc., can solve the problems that cannot be directly converted to the Bacillus family

Pending Publication Date: 2022-03-01
GINKGO BIOWORKS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high genetic diversity among bacteria, findings based on Bacillus subtilis studies often cannot be directly translated to family internal and external members of the Bacillus family

Method used

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  • Targeting sequence for paenibacillus-based endospore display platform
  • Targeting sequence for paenibacillus-based endospore display platform
  • Targeting sequence for paenibacillus-based endospore display platform

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0246] Example 1: General protocol for identifying collagen-like spore surface proteins suitable for endospore display

[0247] Search for ORFs containing collagen-like GXX repeats in the complete genome of Paenibacillus sp. NRRL B-50972. Collagen-like spore surface proteins were then observed by transmission electron microscopy ( figure 1 ). The presence of collagen-like spore surface proteins was also confirmed experimentally by mass spectrometry. Briefly, Paenibacillus NRRL B-50972 spores were trypsinized to remove surface proteins. Spores were removed by centrifugation, and the supernatant was analyzed by mass spectrometry to verify the presence of collagen-like spore surface proteins. This general protocol was used to identify endogenous Paenibacillus sp. NRRL B-50972 proteins with N-terminal targeting sequences identified by SEQ ID NO: 1-10. The same method can be used to identify spore surface proteins and corresponding N-terminal targeting sequences from Chlorobaci...

Embodiment 2

[0248] Example 2: General Protocol for the Preparation of Recombinant Paenibacillus Endospores Displaying Green Fluorescent Protein (GFP)

[0249] To generate the fusion construct, the gene encoding GFP was combined by gene synthesis with a DNA segment encoding the amino acids of the published N-terminal targeting sequence (SEQ ID NO: 1 ) of Paenibacillus sp. NRRL B-50972 (published in fused under the control of the native promoter of the N-terminal targeting sequence) and cloned into the E. coli / Paenibacillus shuttle vector pAP13. The resulting vector construct was introduced into Paenibacillus sp. NRRL B-50972. Correct transformants were then grown in Schaeffer's sporulation broth at 30°C until sporulation. Paenibacillus sp. NRRL B-50972 spores expressing the fusion construct were then examined by epifluorescence microscopy. GFP was visible on spores expressing the fusion construct ( Figure 2A ). Paenibacillus sp. NRRL B-50972 spores were also examined by flow cytometry...

Embodiment 3

[0250] Example 3: General protocol for making recombinant Paenibacillus endospores displaying any protein of interest

[0251] Paenibacillus cells (e.g., Paenibacillus sp. NRRL B-50972) can be cultured, transformed, and screened as described above in Example 2 to produce spores with N-terminal spore surface targeting sequences according to the present disclosure fusion constructs. Screening can be performed by mass spectrometry or any other biochemical or visual means known in the art (eg, the protein of interest can be tagged with GFP or other selection / screening tags). The N-terminal targeting sequence used to generate the fusion construct may comprise a polypeptide of any one of SEQ ID NO: 2, 4, 6, 8 or 10, 18, 20, 21, 22, 24, 26, 28, 30, or fragments or variants thereof. In some aspects, the N-terminal targeting sequence may comprise a residue having one or more residues corresponding to the pairwise alignment of SEQ ID NO: 2 and 8 ( image 3 ) sequence of the same resi...

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Abstract

Signal sequences useful for targeting proteins and peptides to the surface of endophytic spores produced by family members of Paenibacillus and methods of using the same are provided. Also provided are the use of specific N-terminal targeting sequences and derivatives thereof to display heterologous molecules, such as peptides, polypeptides and other recombinant constructs, on the surface of spores of family members of Paenibacillus genus.

Description

[0001] Cross References to Related Applications [0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 848,533, filed May 15, 2019, the entire contents of which are incorporated herein by reference. [0003] Citing an Electronically Submitted Sequence Listing [0004] An official copy of the Sequence Listing is submitted electronically via EFS-Web as a Sequence Listing in ASCII format, named "BCS199006_WO_ST25.txt", created on May 14, 2020, 85 kilobytes in size, and compatible with this The instruction manual is submitted at the same time. The Sequence Listing contained in this document in ASCII format is part of this specification and is incorporated herein by reference in its entirety. technical field [0005] The present disclosure relates generally to endospore display platforms, related display methods, spore surface targeting sequences and fusion protein constructs comprising the same, recombinant endospore compositions, and methods for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/195C12N15/62
CPCC07K14/195C07K2319/035C12N3/00C12N11/16A01N63/25A01N63/50C12R2001/01C12N1/205C12N15/74C12N15/63
Inventor D·柯蒂斯K·蒂普顿
Owner GINKGO BIOWORKS INC
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