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      The art of vector engineering: towards the construction of next‐generation genetic tools

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          Summary

          When recombinant DNA technology was developed more than 40 years ago, no one could have imagined the impact it would have on both society and the scientific community. In the field of genetic engineering, the most important tool developed was the plasmid vector. This technology has been continuously expanding and undergoing adaptations. Here, we provide a detailed view following the evolution of vectors built throughout the years destined to study microorganisms and their peculiarities, including those whose genomes can only be revealed through metagenomics. We remark how synthetic biology became a turning point in designing these genetic tools to create meaningful innovations. We have placed special focus on the tools for engineering bacteria and fungi (both yeast and filamentous fungi) and those available to construct metagenomic libraries. Based on this overview, future goals would include the development of modular vectors bearing standardized parts and orthogonally designed circuits, a task not fully addressed thus far. Finally, we present some challenges that should be overcome to enable the next generation of vector design and ways to address it.

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          Most cited references167

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          Foundations for engineering biology.

          Drew Endy (2005)
          Engineered biological systems have been used to manipulate information, construct materials, process chemicals, produce energy, provide food, and help maintain or enhance human health and our environment. Unfortunately, our ability to quickly and reliably engineer biological systems that behave as expected remains quite limited. Foundational technologies that make routine the engineering of biology are needed. Vibrant, open research communities and strategic leadership are necessary to ensure that the development and application of biological technologies remains overwhelmingly constructive.
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            Golden Gate Shuffling: A One-Pot DNA Shuffling Method Based on Type IIs Restriction Enzymes

            We have developed a protocol to assemble in one step and one tube at least nine separate DNA fragments together into an acceptor vector, with 90% of recombinant clones obtained containing the desired construct. This protocol is based on the use of type IIs restriction enzymes and is performed by simply subjecting a mix of 10 undigested input plasmids (nine insert plasmids and the acceptor vector) to a restriction-ligation and transforming the resulting mix in competent cells. The efficiency of this protocol allows generating libraries of recombinant genes by combining in one reaction several fragment sets prepared from different parental templates. As an example, we have applied this strategy for shuffling of trypsinogen from three parental templates (bovine cationic trypsinogen, bovine anionic trypsinogen and human cationic trypsinogen) each divided in 9 separate modules. We show that one round of shuffling using the 27 trypsinogen entry plasmids can easily produce the 19,683 different possible combinations in one single restriction-ligation and that expression screening of a subset of the library allows identification of variants that can lead to higher expression levels of trypsin activity. This protocol, that we call ‘Golden Gate shuffling’, is robust, simple and efficient, can be performed with templates that have no homology, and can be combined with other shuffling protocols in order to introduce any variation in any part of a given gene.
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              A new efficient gene disruption cassette for repeated use in budding yeast.

              The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.
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                Author and article information

                Contributors
                silvarochar@gmail.com
                Journal
                Microb Biotechnol
                Microb Biotechnol
                10.1111/(ISSN)1751-7915
                MBT2
                Microbial Biotechnology
                John Wiley and Sons Inc. (Hoboken )
                1751-7915
                26 September 2018
                January 2019
                : 12
                : 1 , Thematic Issue on Crystal Ball ( doiID: 10.1111/mbt2.2019.12.issue-1 )
                : 125-147
                Affiliations
                [ 1 ] Ribeirão Preto Medical School University of São Paulo Ribeirão Preto, São Paulo 14049‐900 Brazil
                [ 2 ] School of Philosophy, Science and Letters of Ribeirão Preto University of São Paulo Ribeirão Preto, São Paulo 14049‐900 Brazil
                Author notes
                [*] [* ]For correspondence. E‐mail: silvarochar@ 123456gmail.com ; Tel. (+55) 16 3602 3107; Fax +55 16 3633 6840.
                Author information
                http://orcid.org/0000-0003-1572-2279
                http://orcid.org/0000-0001-6319-631X
                Article
                MBT213318
                10.1111/1751-7915.13318
                6302727
                30259693
                cdc38ec9-ae57-4614-bdc5-1227fa12368a
                © 2018 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

                This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

                History
                : 26 July 2018
                : 29 August 2018
                : 31 August 2018
                Page count
                Figures: 7, Tables: 2, Pages: 23, Words: 17243
                Funding
                Funded by: São Paulo Research Foundation – FAPESP
                Award ID: 2012/22921‐8
                Award ID: 2015/04309‐1
                Award ID: 2016/03763‐3
                Award ID: 2016/05472‐6
                Award ID: 2017/17924‐1
                Award ID: 2016/06323‐4
                Award ID: 2016/19179‐9
                Categories
                Minireview
                Minireviews
                Custom metadata
                2.0
                mbt213318
                January 2019
                Converter:WILEY_ML3GV2_TO_NLMPMC version:version=5.5.4 mode:remove_FC converted:21.12.2018

                Biotechnology
                Biotechnology

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