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.

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 Translesion DNA Synthesis Across Lesions Induced by Oxidative Products of Pyrimidines: An Insight into the Mechanism by Microscale Thermophoresis

2019 ◽  
Vol 20 (20) ◽  
pp. 5012 ◽  
Author(s):  
Ondrej Hrabina ◽  
Viktor Brabec ◽  
Olga Novakova
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Duplexes ◽  
Translesion Dna Synthesis ◽  
Processing Enzymes ◽  
Microscale Thermophoresis ◽  
Oxidative Products ◽  
Dna Processing ◽  
Damaged Dna

Oxidative stress in cells can lead to the accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized nucleotides such as 2’-deoxyribo-5-hydroxyuridin (HdU) and 2’-deoxyribo-5-hydroxymethyluridin (HMdU) can be inserted into DNA during replication and repair. HdU and HMdU have attracted particular interest because they have different effects on damaged-DNA processing enzymes that control the downstream effects of the lesions. Herein, we studied the chemically simulated translesion DNA synthesis (TLS) across the lesions formed by HdU or HMdU using microscale thermophoresis (MST). The thermodynamic changes associated with replication across HdU or HMdU show that the HdU paired with the mismatched deoxyribonucleoside triphosphates disturbs DNA duplexes considerably less than thymidine (dT) or HMdU. Moreover, we also demonstrate that TLS by DNA polymerases across the lesion derived from HdU was markedly less extensive and potentially more mutagenic than that across the lesion formed by HMdU. Thus, DNA polymerization by DNA polymerase η (polη), the exonuclease-deficient Klenow fragment of DNA polymerase I (KF–), and reverse transcriptase from human immunodeficiency virus type 1 (HIV-1 RT) across these pyrimidine lesions correlated with the different stabilization effects of the HdU and HMdU in DNA duplexes revealed by MST. The equilibrium thermodynamic data obtained by MST can explain the influence of the thermodynamic alterations on the ability of DNA polymerases to bypass lesions induced by oxidative products of pyrimidines. The results also highlighted the usefulness of MST in evaluating the impact of oxidative products of pyrimidines on the processing of these lesions by damaged DNA processing enzymes.

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.

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 Engineering polymerases for applications in synthetic biology

2020 ◽  
Vol 53 ◽  
Author(s):  
Ali Nikoomanzar ◽  
Nicholas Chim ◽  
Eric J. Yik ◽  
John C. Chaput
Keyword(s):  
Cell Division ◽  
Synthetic Biology ◽  
Substrate Specificity ◽  
Physicochemical Properties ◽  
Genetic Information ◽  
Biomedical Applications ◽  
Dna Polymerases ◽  
Enzyme Engineering ◽  
Practical Applications ◽  
Naturally Occurring

Abstract DNA polymerases play a central role in biology by transferring genetic information from one generation to the next during cell division. Harnessing the power of these enzymes in the laboratory has fueled an increase in biomedical applications that involve the synthesis, amplification, and sequencing of DNA. However, the high substrate specificity exhibited by most naturally occurring DNA polymerases often precludes their use in practical applications that require modified substrates. Moving beyond natural genetic polymers requires sophisticated enzyme-engineering technologies that can be used to direct the evolution of engineered polymerases that function with tailor-made activities. Such efforts are expected to uniquely drive emerging applications in synthetic biology by enabling the synthesis, replication, and evolution of synthetic genetic polymers with new physicochemical properties.

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.

A rapid and efficient PCR-based mutagenesis method applicable to cell physiology study

2005 ◽  
Vol 288 (6) ◽  
pp. C1273-C1278 ◽  
Author(s):  
Jae-Kyun Ko ◽  
Jianjie Ma
Keyword(s):  
Molecular Biology ◽  
Protein Engineering ◽  
Restriction Enzyme ◽  
Cost Effective ◽  
Restriction Enzymes ◽  
Recognition Site ◽  
Cell Physiology ◽  
Multiple Site ◽  
Functional Studies ◽  
Engineering Studies

PCR-based mutagenesis is a cornerstone of molecular biology and protein engineering studies. Herein we describe a rapid and highly efficient mutagenesis method using type IIs restriction enzymes. A template gene is amplified into two separate PCR fragments using two pairs of anchor and mutagenic primers. Mutated sequences are located near the recognition site of a type IIs restriction enzyme. After digestion of two fragments with a type IIs enzyme, exposed cohesive ends that are complementary to each other are then ligated together to generate a mutated gene. We applied this method to introduce multiple site-directed mutations in EGFP and Bcl-2 family genes and observed perfect mutagenesis efficiency at the desired sites. This efficient and cost-effective mutagenesis method can be applied to a wide variety of structural and functional studies in cell physiology.

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