scholarly journals GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris

2017 ◽  
Vol 11 (1) ◽  
Author(s):  
Roland Prielhofer ◽  
Juan J. Barrero ◽  
Stefanie Steuer ◽  
Thomas Gassler ◽  
Richard Zahrl ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Pichia Pastoris ◽  
Golden Gate ◽  
Yeast Pichia Pastoris ◽  
Cloning System ◽  
Modular Cloning

scholarly journals Peripheral infrastructure vectors and an extended set of plant parts for the modular cloning system

2017 ◽  
Author(s):  
Johannes Gantner ◽  
Theresa Ilse ◽  
Jana Ordon ◽  
Carola Kretschmer ◽  
Ramona Gruetzner ◽  
...  
Keyword(s):  
Building Blocks ◽  
Research Community ◽  
Dna Assembly ◽  
Golden Gate ◽  
Hierarchical Assembly ◽  
Cloning System ◽  
Plant Parts ◽  
Modular Cloning ◽  
Cost Efficient ◽  
Combinatorial Assembly

AbstractStandardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. This combinatorial assembly strategy meets the increasingly complex demands in biotechnology and bioengineering, and also represents a cost-efficient and versatile alternative to previous molecular cloning techniques. For Modular Cloning, a collection of commonly used Plant Parts was previously released together with the Modular Cloning toolkit itself, which largely facilitated the adoption of this cloning system in the research community. Here, a collection of approximately 80 additional phytobricks is provided. These phytobricks comprise e.g. modules for inducible expression systems, different promoters or epitope tags, which will increase the versatility of Modular Cloning-based DNA assemblies. Furthermore, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. Additionally, DNA modules and assembly strategies for connecting Modular Cloning with Gateway Cloning are presented, which may serve as an interface between available resources and newly adopted hierarchical assembly strategies. The presented material will be provided as a toolkit to the plant research community and will further enhance the usefulness and versatility of Modular Cloning.

scholarly journals CyanoGate: A Golden Gate modular cloning suite for engineering cyanobacteria based on the plant MoClo syntax

2018 ◽  
Author(s):  
Ravendran Vasudevan ◽  
Grant A.R. Gale ◽  
Alejandra A. Schiavon ◽  
Anton Puzorjov ◽  
John Malm ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Plasmid Vector ◽  
Dna Assembly ◽  
Golden Gate ◽  
Biotechnological Applications ◽  
Genomic Information ◽  
Exponential Increase ◽  
Multigene Expression ◽  
Modular Cloning ◽  
Biology Research

ABSTRACTRecent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and exchange of parts between different labs and biological systems. However, a standardised Modular Cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential in biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. We have generated a suite of parts and acceptor vectors for making i) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and ii) transient multigene expression and repression systems using known and novel replicative vectors. We have tested and compared the CyanoGate system in the established model cyanobacteriumSynechocystissp. PCC 6803 and the more recently described fast-growing strainSynechococcus elongatusUTEX 2973. The system is publicly available and can be readily expanded to accommodate other standardised MoClo parts.

scholarly journals Komagataeibacter tool kit (KTK): a modular cloning system for multigene constructs and programmed protein secretion from cellulose producing bacteria

2021 ◽  
Author(s):  
Vivianne J Goosens ◽  
Kenneth T Walker ◽  
Silvia M Aragon ◽  
Amritpal Singh ◽  
Vivek R Senthivel ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Protein Secretion ◽  
Functional Expression ◽  
Dna Assembly ◽  
Cloning System ◽  
Pure Cellulose ◽  
Modular Cloning ◽  
High Yields ◽  
Short Time ◽  
Cellulose Materials

Bacteria proficient at producing cellulose are an attractive synthetic biology host for the emerging field of Engineered Living Materials (ELMs). Species from the Komagataeibacter genus produce high yields of pure cellulose materials in a short time with minimal resources, and pioneering work has shown that genetic engineering in these strains is possible and can be used to modify the material and its production. To accelerate synthetic biology progress in these bacteria, we introduce here the Komagataeibacter tool kit (KTK), a standardised modular cloning system based on Golden Gate DNA assembly that allows DNA parts to be combined to build complex multigene constructs expressed in bacteria from plasmids. Working in Komagataeibacter rhaeticus, we describe basic parts for this system, including promoters, fusion tags and reporter proteins, before showcasing how the assembly system enables more complex designs. Specifically, we use KTK cloning to reformat the Escherichia coli curli amyloid fibre system for functional expression in K. rhaeticus, and go on to modify it as a system for programming protein secretion from the cellulose producing bacteria. With this toolkit, we aim to accelerate modular synthetic biology in these bacteria, and enable more rapid progress in the emerging ELMs community.

scholarly journals Joint Universal Modular Plasmids (JUMP): A flexible and comprehensive platform for synthetic biology

2019 ◽  
Author(s):  
Marcos Valenzuela-Ortega ◽  
Christopher French
Keyword(s):  
Synthetic Biology ◽  
Golden Gate ◽  
Flexible Design ◽  
Test Cycle ◽  
Design Build ◽  
Modular Cloning ◽  
Multiple Cloning Sites ◽  
Golden Gate Cloning

AbstractComplex multi-gene plasmids can be built from basic DNA parts in a reliable and automation friendly way using modular cloning standards, based on Golden Gate cloning. However, each toolkit or standard is limited to one or a few different vectors, which has led to an overabundance of toolkits with varying degrees of compatibility. Here, we present the Joint Universal Modular Plasmids (JUMP), a vector design that overcomes the limitations of current toolkits by expanding the paradigm of modular cloning: all vectors can be modified using modular cloning in an orthogonal way using multiple cloning sites. This allows researchers to introduce any feature into any JUMP vector and simplifies the Design-Build-Test cycle of synthetic biology. JUMP vectors are compatible with PhytoBrick basic parts, BioBricks and the Registry of Standard Biological Parts, and the Standard European Vector Architecture (SEVA). Due to their flexible design, JUMP vectors have the potential to be a universal platform for synthetic biology regardless of host and application. A collection of JUMP backbones and microbial PhytoBrick basic parts are available for distribution.

scholarly journals Adsorption process and mechanism of heavy metal ions by different components of cells, using yeast (Pichia pastoris) and Cu2+ as biosorption models

RSC Advances ◽  
2021 ◽  
Vol 11 (28) ◽  
pp. 17080-17091
Author(s):  
Xinggang Chen ◽  
Zhuang Tian ◽  
Haina Cheng ◽  
Gang Xu ◽  
Hongbo Zhou
Keyword(s):  
Heavy Metal ◽  
Pichia Pastoris ◽  
Metal Ions ◽  
Cell Walls ◽  
Heavy Metal Ions ◽  
Adsorption Process ◽  
Yeast Pichia Pastoris ◽  
Adsorption Sites

The Cu2+ first bound to the outer mannan and finally entered the cytoplasm. During the whole adsorption process, the number of adsorption sites in the outer and middle cell walls was the largest, and then gradually decreased.

The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris

2014 ◽  
Vol 1841 (2) ◽  
pp. 215-226 ◽  
Author(s):  
Lisa Klug ◽  
Pablo Tarazona ◽  
Clemens Gruber ◽  
Karlheinz Grillitsch ◽  
Brigitte Gasser ◽  
...  
Keyword(s):  
Pichia Pastoris ◽  
Methylotrophic Yeast ◽  
Yeast Pichia Pastoris ◽  
Methylotrophic Yeast Pichia Pastoris

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 Refactoring of a synthetic raspberry ketone pathway with EcoFlex

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Simon J. Moore ◽  
Yonek B. Hleba ◽  
Sarah Bischoff ◽  
David Bell ◽  
Karen M. Polizzi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
Combinatorial Libraries ◽  
Value Added ◽  
Microtiter Plate ◽  
Fine Tuning ◽  
Golden Gate ◽  
E Coli ◽  
Fine Chemical ◽  
Raspberry Ketone

Abstract Background  A key focus of synthetic biology is to develop microbial or cell-free based biobased routes to value-added chemicals such as fragrances. Originally, we developed the EcoFlex system, a Golden Gate toolkit, to study genes/pathways flexibly using Escherichia coli heterologous expression. In this current work, we sought to use EcoFlex to optimise a synthetic raspberry ketone biosynthetic pathway. Raspberry ketone is a high-value (~ £20,000 kg−1) fine chemical farmed from raspberry (Rubeus rubrum) fruit. Results  By applying a synthetic biology led design-build-test-learn cycle approach, we refactor the raspberry ketone pathway from a low level of productivity (0.2 mg/L), to achieve a 65-fold (12.9 mg/L) improvement in production. We perform this optimisation at the prototype level (using microtiter plate cultures) with E. coli DH10β, as a routine cloning host. The use of E. coli DH10β facilitates the Golden Gate cloning process for the screening of combinatorial libraries. In addition, we also newly establish a novel colour-based phenotypic screen to identify productive clones quickly from solid/liquid culture. Conclusions  Our findings provide a stable raspberry ketone pathway that relies upon a natural feedstock (L-tyrosine) and uses only constitutive promoters to control gene expression. In conclusion we demonstrate the capability of EcoFlex for fine-tuning a model fine chemical pathway and provide a range of newly characterised promoter tools gene expression in E. coli.

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