scholarly journals Engineering a Minimal 1185 Bp Cloning Vector from a Puc18 Plasmid Backbone with an Extended Multiple Cloning Site

2018 ◽  
Author(s):  
Jens Staal ◽  
Wouter De Schamphelaire ◽  
Rudi Beyaert
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Restriction Enzyme ◽  
Essential Role ◽  
Building Blocks ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Unique Restriction ◽  
Plasmid Backbone

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of a ~1kb fully functional cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

scholarly journals Engineering a Minimal Cloning Vector from a pUC18 Plasmid Backbone with an Extended Multiple Cloning Site

2018 ◽  
Author(s):  
Jens Staal ◽  
Wouter De Schamphelaire ◽  
Rudi Beyaert
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Restriction Enzyme ◽  
Essential Role ◽  
Building Blocks ◽  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Unique Restriction ◽  
Plasmid Backbone

Minimal plasmids play an essential role in many intermediate steps in molecular biology. They can for example be used to assemble building blocks in synthetic biology or be used as intermediate cloning plasmids that are ideal for PCR-based mutagenesis methods. A small backbone also opens up for additional unique restriction enzyme cloning sites. Here we describe the generation of pICOz, a 1185 bp fully functional high-copy cloning plasmid with an extended multiple cloning site (MCS). To our knowledge, this is the smallest high-copy cloning vector ever described.

scholarly journals Engineering a minimal cloning vector from a pUC18 plasmid backbone with an extended multiple cloning site

BioTechniques ◽  
2019 ◽  
Vol 66 (6) ◽  
pp. 254-259 ◽  
Author(s):  
Jens Staal ◽  
Kübra Alci ◽  
Wouter De Schamphelaire ◽  
Martine Vanhoucke ◽  
Rudi Beyaert
Keyword(s):  
Cloning Vector ◽  
Multiple Cloning Site ◽  
Plasmid Backbone

Molecular Biology

2016 ◽  
Author(s):  
Aysha Divan ◽  
Janice Royds
Keyword(s):  
Synthetic Biology ◽  
Personalized Medicine ◽  
Molecular Biology ◽  
Dna Fingerprinting ◽  
Genetically Modified Organisms ◽  
Building Blocks ◽  
New Drugs ◽  
Human History ◽  
Short Introduction ◽  
Wide Range

Molecular biology is the story of the molecules of life, their relationships, and how these interactions are controlled. Its applications are wide and growing; the power of molecular biology can now be harnessed to treat diseases, solve crimes, map human history, and produce genetically modified organisms and crops. Starting with the building blocks established by Darwin, Wallace, and Mendel, and the discovery of the structure of DNA in 1953, Molecular Biology: A Very Short Introduction considers the wide range of applications for molecular biology today, including the development of new drugs and DNA fingerprinting, and looks forward to two key areas of evolving research: personalized medicine and synthetic biology.

scholarly journals A simple and general approach to control the activity of DNA processing enzymes

2021 ◽  
Author(s):  
Merve-Zeynep Kesici ◽  
Philip Tinnefeld ◽  
Andres M Vera
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Restriction Enzyme ◽  
Dna Polymerases ◽  
Endonuclease Activity ◽  
Processing Enzymes ◽  
Hot Start ◽  
Comparable Performance ◽  
General Method ◽  
Dna Processing

DNA processing enzymes, such as DNA polymerases and endonucleases, have found many applications in biotechnology, molecular diagnostics, and synthetic biology, among others. The development of enzymes with controllable activity, such as hot-start or light-activatable versions, has boosted their applications and improved the sensitivity and specificity of the existing ones. However, current approaches to produce controllable enzymes are experimentally demanding to develop and case specific. Here, we introduce a simple and general method to design light-start DNA processing enzymes. In order to prove its versatility, we applied our method to three DNA polymerases commonly used in biotechnology, including the Phi29 (mesophilic), Taq and Pfu polymerases, and one restriction enzyme. Light-start enzymes showed suppressed polymerase, exonuclease and endonuclease activity until they were re-activated by an UV pulse. Finally, we applied our enzymes to common molecular biology assays, and showed comparable performance to commercial hot-start enzymes.

Probing the Substrate Promiscuity of Isopentenyl Phosphate Kinase as a Platform for Hemiterpene Analogue Production

2019 ◽  
Author(s):  
Sean Lund ◽  
Taylor Courtney ◽  
Gavin Williams
Keyword(s):  
Synthetic Biology ◽  
Building Blocks ◽  
Isoprenoid Biosynthesis ◽  
Thermoplasma Acidophilum ◽  
Substrate Promiscuity ◽  
Enzymatic Pathways ◽  
First Time ◽  
Rate Limiting ◽  
Alcohol Dependent ◽  
Isopentenyl Phosphate

Isoprenoids are a large class of natural products with wide-ranging applications. Synthetic biology approaches to the manufacture of isoprenoids and their new-to-nature derivatives are limited due to the provision in Nature of just two hemiterpene building blocks for isoprenoid biosynthesis. To address this limitation, artificial chemo-enzymatic pathways such as the alcohol-dependent hemiterpene pathway (ADH) serve to leverage consecutive kinases to convert exogenous alcohols to pyrophosphates that could be coupled to downstream isoprenoid biosynthesis. To be successful, each kinase in this pathway should be permissive of a broad range of substrates. For the first time, we have probed the promiscuity of the second enzyme in the ADH pathway, isopentenyl phosphate kinase from Thermoplasma acidophilum, towards a broad range of acceptor monophosphates. Subsequently, we evaluate the suitability of this enzyme to provide non-natural pyrophosphates and provide a critical first step in characterizing the rate limiting steps in the artificial ADH pathway.<br>

A Redesigned Planet?

2020 ◽  
pp. 234-296
Author(s):  
John Parrington
Keyword(s):  
Stem Cell ◽  
Synthetic Biology ◽  
Molecular Biology ◽  
Medical Research ◽  
Genome Editing ◽  
New Technologies ◽  
Ethical Issues ◽  
Biological Weapons ◽  
New Development ◽  
Whole Number

Given the speed of change in the development of new technologies mentioned in this book such as genome editing, optogenetics, stem cell organoids, and synthetic biology, it is hard to predict exactly how radically these technologies are likely to transform our lives in coming decades. What is clear is that as exciting as the new biotechnologies are in terms of their impact on medical research, medicine, and agriculture, they also raise a whole number of socio-political and ethical issues. These include concerns about whether monkeys engineered to have genetic similarities to humans might lead to a ‘Planet of the Apes’ scenario, and fears about ‘designer babies’ being produced in the future to have greater beauty, intelligence or sporting skill. Although one potentially positive new development is the rise of a ‘biohacker’ movement which seeks to make molecular biology more accessible to ordinary people, there are also fears that in the wrong hands genome editing might be used to create new types of biological weapons for terrorist organisations. While such fears should not be dismissed as just an overreaction, to some extent they rest on an underestimation of the complexity of the Iink between the human genome and looks, intelligence, and sporting ability, or of the difficulties involved in creating a deadly virus that is worse than naturally occurring ones. Ultimately, the best way to ensure that new technologies are used for human benefit, not harm, is to take part in an informed debate and use public lobbying to argue for them to be developed safely, ethically, and responsibly.

Life as It Could Be

2020 ◽  
pp. 101-114
Author(s):  
Luis Campos
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
The Future ◽  
Forms Of Life ◽  
Life On Earth ◽  
The Universe

This chapter explores the intersection between two related fields: synthetic biology and astrobiology. Pushing the engineering of life past traditional limits in molecular biology and expanding the envelope of life to forms never before extant, synthetic biologists are now beginning to design experimental ways of getting at what astrobiologists have long suspected: that the life known here on Earth is but a subset of vast combinatorial possibilities in the universe. The resonances between the future engineered possibilities of this world and speculations about possible biologies on habitable others are not merely happenstance. Indeed, there is a curious and compelling deeper history interlinking scientific speculation about new forms of life elsewhere in the universe with visions for the human-directed engineering of new forms of life on Earth. For decades, the astrobiological and the synthetic biological have mutually inspired each other and overlapped in powerful genealogical ways.

scholarly journals Synthetic Biology Open Language (SBOL) Version 2.1.0

2016 ◽  
Vol 13 (3) ◽  
Author(s):  
Jacob Beal ◽  
Robert Sidney Cox ◽  
Raik Grünberg ◽  
James McLaughlin ◽  
Tramy Nguyen ◽  
...  
Keyword(s):  
Metabolic Engineering ◽  
Synthetic Biology ◽  
Molecular Biology ◽  
Best Practices ◽  
Basic Sequence ◽  
Special Issue ◽  
Biological Design ◽  
Version 2.0 ◽  
Topology Information ◽  
Synthetic Biology Open Language

SummarySynthetic biology builds upon the techniques and successes of genetics, molecular biology, and metabolic engineering by applying engineering principles to the design of biological systems. The field still faces substantial challenges, including long development times, high rates of failure, and poor reproducibility. One method to ameliorate these problems would be to improve the exchange of information about designed systems between laboratories. The Synthetic Biology Open Language (SBOL) has been developed as a standard to support the specification and exchange of biological design information in synthetic biology, filling a need not satisfied by other pre-existing standards. This document details version 2.1 of SBOL that builds upon version 2.0 published in last year’s JIB special issue. In particular, SBOL 2.1 includes improved rules for what constitutes a valid SBOL document, new role fields to simplify the expression of sequence features and how components are used in context, and new best practices descriptions to improve the exchange of basic sequence topology information and the description of genetic design provenance, as well as miscellaneous other minor improvements.

scholarly journals Construction of membrane-bound artificial cells using microfluidics: a new frontier in bottom-up synthetic biology

2016 ◽  
Vol 44 (3) ◽  
pp. 723-730 ◽  
Author(s):  
Yuval Elani
Keyword(s):  
Synthetic Biology ◽  
High Throughput ◽  
Lipid Membranes ◽  
Spatial Organization ◽  
Building Blocks ◽  
Artificial Cells ◽  
Bottom Up ◽  
Grand Challenges ◽  
New Frontier ◽  
Membrane Bound

The quest to construct artificial cells from the bottom-up using simple building blocks has received much attention over recent decades and is one of the grand challenges in synthetic biology. Cell mimics that are encapsulated by lipid membranes are a particularly powerful class of artificial cells due to their biocompatibility and the ability to reconstitute biological machinery within them. One of the key obstacles in the field centres on the following: how can membrane-based artificial cells be generated in a controlled way and in high-throughput? In particular, how can they be constructed to have precisely defined parameters including size, biomolecular composition and spatial organization? Microfluidic generation strategies have proved instrumental in addressing these questions. This article will outline some of the major principles underpinning membrane-based artificial cells and their construction using microfluidics, and will detail some recent landmarks that have been achieved.

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