scholarly journals Construction of multicellular yeast networks using the communication toolkit with variable specificity and attenuation

2021 ◽  
Author(s):  
Nicolas Krink ◽  
Anne Christina Loechner ◽  
Alexander Anders ◽  
Joerg Kahnt ◽  
Georg Hochberg ◽  
...  
Keyword(s):  
Cellular Network ◽  
Yeast Species ◽  
Logic Gate ◽  
Cell Communication ◽  
Golden Gate ◽  
Pheromone Receptor ◽  
Inducible Promoters ◽  
Network Engineering ◽  
Fungal Mating ◽  
Golden Gate Cloning

The key next step in synthetic biology is to extend cellular network engineering to the multicellular level by utilizing cell-cell communication for information processing. To facilitate the implementation of multicellular networks in the most commonly used eukaryotic chassis, Saccharomyces cerevisiae, we developed the yeast communication toolkit (YCTK). This toolkit is based on the fungal mating pathway and contains five pheromone-inducible promoters (response parts), eleven pheromones (α-factors; sender parts), eleven pheromone receptors (Ste2; receiver parts), as well as five Bar1 proteases (suppressor parts). All YCTK parts were thoroughly characterized and are compatible with the commonly used yeast Golden Gate cloning standard. We demonstrated the application of the YCTK by implementing several different logic gate-like population networks. Furthermore, we used this toolkit to investigate the pheromone-receptor promiscuity patterns among different yeast species. This toolkit extends currently available resources for construction of complex multicellular eukaryotic networks with varying degrees of promiscuity and attenuation.

scholarly journals Joint universal modular plasmids (JUMP): a flexible vector platform for synthetic biology

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Copy Number ◽  
Life Science ◽  
Insertion Site ◽  
Golden Gate ◽  
Common Elements ◽  
Modern Life ◽  
Specific Host ◽  
Modular Cloning ◽  
Golden Gate Cloning ◽  
Selection Of

Abstract Generation of new DNA constructs is an essential process in modern life science and biotechnology. Modular cloning systems based on Golden Gate cloning, using Type IIS restriction endonucleases, allow assembly of complex multipart constructs from reusable basic DNA parts in a rapid, reliable and automation-friendly way. Many such toolkits are available, with varying degrees of compatibility, most of which are aimed at specific host organisms. Here, we present a vector design which allows simple vector modification by using modular cloning to assemble and add new functions in secondary sites flanking the main insertion site (used for conventional modular cloning). Assembly in all sites is compatible with the PhytoBricks standard, and vectors are compatible with the Standard European Vector Architecture (SEVA) as well as BioBricks. We demonstrate that this facilitates the construction of vectors with tailored functions and simplifies the workflow for generating libraries of constructs with common elements. We have made available a collection of vectors with 10 different microbial replication origins, varying in copy number and host range, and allowing chromosomal integration, as well as a selection of commonly used basic parts. This design expands the range of hosts which can be easily modified by modular cloning and acts as a toolkit which can be used to facilitate the generation of new toolkits with specific functions required for targeting further hosts.

scholarly journals Golden Mutagenesis: An efficient multi-sitesaturation mutagenesis approach by Golden Gate cloning with automated primer design

2018 ◽  
Author(s):  
Pascal Püllmann ◽  
Chris Ulpinnis ◽  
Sylvestre Marillonnet ◽  
Ramona Gruetzner ◽  
Steffen Neumann ◽  
...  
Keyword(s):  
Web Application ◽  
Pcr Amplification ◽  
Restriction Enzymes ◽  
Primer Design ◽  
Golden Gate ◽  
Efficient Tool ◽  
Multiple Gene ◽  
Reading Frame ◽  
Cloning Technique ◽  
Golden Gate Cloning

Site-directed methods for the generation of genetic diversity are essential tools in the field of directed enzyme evolution. The Golden Gate cloning technique has been proven to be an efficient tool for a variety of cloning setups. The utilization of restriction enzymes which cut outside of their recognition domain allows the assembly of multiple gene fragments obtained by PCR amplification without altering the open reading frame of the reconstituted gene. We have developed a protocol, termed Golden Muta-genesis that allows the rapid, straightforward, reliable and inexpensive construction of mutagenesis libraries. One to five amino acid positions within a coding sequence could be altered simultaneously using a protocol which can be performed within one day. To facilitate the implementation of this technique, a software library and web application for automated primer design and for the graphical evaluation of the randomization success based on the sequencing results was developed. This allows facile primer design and application of Golden Mutagenesis also for laboratories, which are not specialized in molecular biology.

scholarly journals Construction of Multiple Guide RNAs in CRISPR/Cas9 Vector Using Stepwise or Simultaneous Golden Gate Cloning: Case Study for Targeting the FAD2 and FATB Multigene in Soybean

Plants ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 2542
Author(s):  
Won-Nyeong Kim ◽  
Hye-Jeong Kim ◽  
Young-Soo Chung ◽  
Hyun-Uk Kim
Keyword(s):  
Reverse Genetics ◽  
Restriction Enzymes ◽  
Golden Gate ◽  
Knock Out ◽  
Guide Rnas ◽  
Genetics Research ◽  
Multiple Genes ◽  
Cas9 Protein ◽  
Cloning Method ◽  
Golden Gate Cloning

CRISPR/Cas9 is a commonly used technique in reverse-genetics research to knock out a gene of interest. However, when targeting a multigene family or multiple genes, it is necessary to construct a vector with multiple single guide RNAs (sgRNAs) that can navigate the Cas9 protein to the target site. In this protocol, the Golden Gate cloning method was used to generate multiple sgRNAs in the Cas9 vector. The vectors used were pHEE401E_UBQ_Bar and pBAtC_tRNA, which employ a one-promoter/one-sgRNA and a polycistronic-tRNA-gRNA strategy, respectively. Golden Gate cloning was performed with type IIS restriction enzymes to generate gRNA polymers for vector inserts. Four sgRNAs containing the pHEE401E_UBQ_Bar vector and four to six sgRNAs containing the pBAtC_tRNA vector were constructed. In practice, we constructed multiple sgRNAs targeting multiple genes of FAD2 and FATB in soybean using this protocol. These three vectors were transformed into soybeans using the Agrobacterium-mediated method. Using deep sequencing, we confirmed that the T0 generation transgenic soybean was edited at various indel ratios in the predicted target regions of the FAD2 and FATB multigenes. This protocol is a specific guide that allows researchers to easily follow the cloning of multiple sgRNAs into commonly used CRISPR/Cas9 vectors for plants.

Facile generation of antibody heavy and light chain diversities for yeast surface display by Golden Gate Cloning

2019 ◽  
Vol 400 (3) ◽  
pp. 383-393 ◽  
Author(s):  
Lukas Roth ◽  
Julius Grzeschik ◽  
Steffen C. Hinz ◽  
Stefan Becker ◽  
Lars Toleikis ◽  
...  
Keyword(s):  
Light Chain ◽  
Surface Display ◽  
Combinatorial Libraries ◽  
Yeast Surface Display ◽  
Golden Gate ◽  
Yeast Display ◽  
Step Method ◽  
One Step ◽  
Yeast Surface ◽  
Golden Gate Cloning

Abstract Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning (GGC) with the conventional three-step method of individual heavy- and light-chain sub-library construction followed by chain combination via yeast mating (YM). We demonstrate that the GGC-based one-step process delivers libraries and antibodies from heavy- and light-chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.

scholarly journals Fast track assembly of multigene constructs using Golden Gate cloning and the MoClo system

Bioengineered ◽  
2012 ◽  
Vol 3 (1) ◽  
pp. 38-43 ◽  
Author(s):  
Stefan Werner ◽  
Carola Engler ◽  
Ernst Weber ◽  
Ramona Gruetzner ◽  
Sylvestre Marillonnet
Keyword(s):  
Fast Track ◽  
Golden Gate ◽  
Golden Gate Cloning

scholarly journals A modular two yeast species secretion system for the production and preparative application of unspecific peroxygenases

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Pascal Püllmann ◽  
Anja Knorrscheidt ◽  
Judith Münch ◽  
Paul R. Palme ◽  
Wolfgang Hoehenwarter ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
High Throughput Screening ◽  
Yeast Species ◽  
Secretion System ◽  
Signal Peptides ◽  
Golden Gate ◽  
Preparative Scale ◽  
One Step ◽  
Protein Tags ◽  
Enzyme Class

AbstractFungal unspecific peroxygenases (UPOs) represent an enzyme class catalysing versatile oxyfunctionalisation reactions on a broad substrate scope. They are occurring as secreted, glycosylated proteins bearing a haem-thiolate active site and rely on hydrogen peroxide as the oxygen source. However, their heterologous production in a fast-growing organism suitable for high throughput screening has only succeeded once—enabled by an intensive directed evolution campaign. We developed and applied a modular Golden Gate-based secretion system, allowing the first production of four active UPOs in yeast, their one-step purification and application in an enantioselective conversion on a preparative scale. The Golden Gate setup was designed to be universally applicable and consists of the three module types: i) signal peptides for secretion, ii) UPO genes, and iii) protein tags for purification and split-GFP detection. The modular episomal system is suitable for use in Saccharomyces cerevisiae and was transferred to episomal and chromosomally integrated expression cassettes in Pichia pastoris. Shake flask productions in Pichia pastoris yielded up to 24 mg/L secreted UPO enzyme, which was employed for the preparative scale conversion of a phenethylamine derivative reaching 98.6 % ee. Our results demonstrate a rapid, modular yeast secretion workflow of UPOs yielding preparative scale enantioselective biotransformations.

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