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Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining

One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and...

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Autores principales: Suárez, Gabriel A, Dugan, Kyle R, Renda, Brian A, Leonard, Sean P, Gangavarapu, Lakshmi Suryateja, Barrick, Jeffrey E
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Oxford University Press 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192602/
https://www.ncbi.nlm.nih.gov/pubmed/32232367
http://dx.doi.org/10.1093/nar/gkaa204
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author Suárez, Gabriel A
Dugan, Kyle R
Renda, Brian A
Leonard, Sean P
Gangavarapu, Lakshmi Suryateja
Barrick, Jeffrey E
author_facet Suárez, Gabriel A
Dugan, Kyle R
Renda, Brian A
Leonard, Sean P
Gangavarapu, Lakshmi Suryateja
Barrick, Jeffrey E
author_sort Suárez, Gabriel A
collection PubMed
description One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism.
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spelling pubmed-71926022020-05-06 Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining Suárez, Gabriel A Dugan, Kyle R Renda, Brian A Leonard, Sean P Gangavarapu, Lakshmi Suryateja Barrick, Jeffrey E Nucleic Acids Res Synthetic Biology and Bioengineering One goal of synthetic biology is to improve the efficiency and predictability of living cells by removing extraneous genes from their genomes. We demonstrate improved methods for engineering the genome of the metabolically versatile and naturally transformable bacterium Acinetobacter baylyi ADP1 and apply them to a genome streamlining project. In Golden Transformation, linear DNA fragments constructed by Golden Gate Assembly are directly added to cells to create targeted deletions, edits, or additions to the chromosome. We tested the dispensability of 55 regions of the ADP1 chromosome using Golden Transformation. The 18 successful multiple-gene deletions ranged in size from 21 to 183 kb and collectively accounted for 23.4% of its genome. The success of each multiple-gene deletion attempt could only be partially predicted on the basis of an existing collection of viable ADP1 single-gene deletion strains and a new transposon insertion sequencing (Tn-Seq) dataset that we generated. We further show that ADP1’s native CRISPR/Cas locus is active and can be retargeted using Golden Transformation. We reprogrammed it to create a CRISPR-Lock, which validates that a gene has been successfully removed from the chromosome and prevents it from being reacquired. These methods can be used together to implement combinatorial routes to further genome streamlining and for more rapid and assured metabolic engineering of this versatile chassis organism. Oxford University Press 2020-05-07 2020-03-30 /pmc/articles/PMC7192602/ /pubmed/32232367 http://dx.doi.org/10.1093/nar/gkaa204 Text en © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Synthetic Biology and Bioengineering
Suárez, Gabriel A
Dugan, Kyle R
Renda, Brian A
Leonard, Sean P
Gangavarapu, Lakshmi Suryateja
Barrick, Jeffrey E
Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
title Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
title_full Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
title_fullStr Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
title_full_unstemmed Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
title_short Rapid and assured genetic engineering methods applied to Acinetobacter baylyi ADP1 genome streamlining
title_sort rapid and assured genetic engineering methods applied to acinetobacter baylyi adp1 genome streamlining
topic Synthetic Biology and Bioengineering
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7192602/
https://www.ncbi.nlm.nih.gov/pubmed/32232367
http://dx.doi.org/10.1093/nar/gkaa204
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