MoCloFlex: A Modular Yet Flexible Cloning System

Klein, Carlo; Emde, Leonie; Kuijpers, Aaron; Sobetzko, Patrick

doi:10.3389/fbioe.2019.00271

Cited by 8 publications

(22 citation statements)

References 32 publications

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“…This fact might require elimination of incompatible restriction sites if parts from different toolboxes are to be used in higher level plasmids. In addition to the standard Phytobrick fusion sites, we designed novel fusion sites for Marburg Collection-specific part categories, namely ORI, AR and 5’ and 3’ connector parts, as described previously (27). To facilitate standard cloning workflows, we defined eight level 0 part categories required for the construction of a level 1 plasmid (Figure 1B).…”

Section: Resultsmentioning

confidence: 99%

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications inVibrio natriegens

Stukenberg

Hensel

Hoff

et al. 2021

Preprint

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Vibrio natriegens is known as the world's fastest growing organism with a doubling time of less than 10 minutes. This incredible growth speed empowers V. natriegens as a chassis for synthetic and molecular biology, potentially replacing E. coli in many applications. While first genetic parts have been built and tested for V. natriegens, a comprehensive toolkit containing well-characterized and standardized parts did not exist. To close this gap, we created the Marburg Collection - a highly flexible Golden Gate Assembly-based cloning toolbox optimized for the emerging chassis organism V. natriegens. The Marburg Collection overcomes the paradigm of plasmid construction - integrating inserts into a backbone - by enabling the de novo assembly of plasmids from basic genetic parts. This allows users to select the plasmid replication origin and resistance part independently, which is highly advantageous when limited knowledge about the behavior of those parts in the target organism is available. Additional design highlights of the Marburg Collection are novel connector parts, which facilitate modular circuit assembly and, optionally, the inversion of individual transcription units to reduce transcriptional crosstalk in multigene constructs. To quantitatively characterize the genetic parts contained in the Marburg Collection in V. natriegens, we developed a reliable microplate reader measurement workflow for reporter experiments and overcame organism-specific challenges. We think the Marburg Collection with its thoroughly characterized parts will provide a valuable resource for the growing V. natriegens community.

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Section: Resultsmentioning

confidence: 99%

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications inVibrio natriegens

Stukenberg

Hensel

Hoff

et al. 2021

Preprint

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“…MoCloFlex, a new modular cloning system for flexible de novo part/plasmid assembly, has been developed, and it has been revealed that it can be used to plan, build, and isolate a custom plasmid within 24 h. This leads to reduced costs and time consumption (Klein et al, 2019). In one study, it was reported that the E. coli "TatExpress" strain resulted in the delivery of 5.4 g/l of human growth hormone to the periplasm by the Tat pathway using fed-batch fermentation.…”

Section: Escherichia Colimentioning

confidence: 99%

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

369

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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“…2A). Additionally, the INS entry vector is level 1 MoClo 19 , MoCloFlex 20 and Marburg Collection 22 compatible. Hence, level 1 transcription units build with the MoClo-System or Marburg Collection as well as larger assemblies build with MoCloFlex can be cloned into the INS entry vector.…”

Section: Modular Assembly Of the Pswap Plasmidmentioning

confidence: 99%

“…A standard assembly of pSwap comprises 7 parts. With a growing number of parts in a golden gate reaction, the number of correctly assembled plasmids decrease 20 . The selection for a correctly assembled pSwap is guided by co-expression of the deoxyviolacein pathway, whose individual genes are distributed among the parts T1, T2, H1α and HA.…”

Section: Modular Assembly Of the Pswap Plasmidmentioning

confidence: 99%

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CRISPR SWAPnDROP -- A multifunctional system for genome editing and large-scale interspecies gene transfer

Teufel,

Klein,

Mager

et al. 2021

Preprint

Self Cite

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The need for diverse chromosomal modifications in biotechnology, synthetic biology and basic research requires the development of new technologies. With CRISPR SWAPnDROP, we extend the limits of genome editing to large-scale in-vivo DNA transfer between bacterial species. Its modular platform approach facilitates species specific adaptation to confer genome editing in various species. In this study, we show the implementation of the CRISPR SWAPnDROP concept for the model organism Escherichia coli and the currently fastest growing and biotechnologically relevant organism Vibrio natriegens. We demonstrate the excision, transfer and integration of 151kb chromosomal DNA between E. coli strains and from E. coli to V. natriegens without size-limiting intermediate DNA extraction. With the transfer of the E. coli MG1655 wild type lac operon, we establish a functional lactose and galactose degradation pathway in V. natriegens to extend its biotechnological spectrum. We also transfer the E. coli DH5α lac operon and make V. natriegens capable of α-complementation -a step towards an ultra-fast cloning strain. Furthermore, CRISPR SWAPnDROP is designed to be the swiss army knife of genome engineering. Its spectrum of application comprises scarless, marker-free, iterative and parallel insertions and deletions, genome rearrangements, as well as gene transfer between strains and across species. The modular character facilitates DNA library applications and the recycling of standardized parts. Its novel multi-color scarless co-selection system significantly improves editing efficiency to 92% for single edits and 83% for quadruple edits and provides visual quality controls throughout the assembly and editing process.

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MoCloFlex: A Modular Yet Flexible Cloning System

Cited by 8 publications

References 32 publications

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications inVibrio natriegens

The Marburg Collection: A Golden Gate DNA Assembly Framework for Synthetic Biology Applications inVibrio natriegens

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

CRISPR SWAPnDROP -- A multifunctional system for genome editing and large-scale interspecies gene transfer

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