scholarly journals Engineering orthogonal dual transcription factors for multi-input synthetic promoters

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Andreas K. Brödel ◽  
Alfonso Jaramillo ◽  
Mark Isalan
Keyword(s):  
Transcription Factors ◽  
Synthetic Biology ◽  
Directed Evolution ◽  
Logic Gates ◽  
Bacteriophage Λ ◽  
Gene Circuits ◽  
Synthetic Promoters ◽  
Core Set ◽  
Artificial Gene ◽  
Current Engineering

Abstract Synthetic biology has seen an explosive growth in the capability of engineering artificial gene circuits from transcription factors (TFs), particularly in bacteria. However, most artificial networks still employ the same core set of TFs (for example LacI, TetR and cI). The TFs mostly function via repression and it is difficult to integrate multiple inputs in promoter logic. Here we present to our knowledge the first set of dual activator-repressor switches for orthogonal logic gates, based on bacteriophage λ cI variants and multi-input promoter architectures. Our toolkit contains 12 TFs, flexibly operating as activators, repressors, dual activator–repressors or dual repressor–repressors, on up to 270 synthetic promoters. To engineer non cross-reacting cI variants, we design a new M13 phagemid-based system for the directed evolution of biomolecules. Because cI is used in so many synthetic biology projects, the new set of variants will easily slot into the existing projects of other groups, greatly expanding current engineering capacities.

scholarly journals Engineering of Synthetic Transcriptional Switches in Yeast

2021 ◽  
Author(s):  
Masahiro Tominaga ◽  
Akihiko Kondo ◽  
Jun Ishii
Keyword(s):  
Transcription Factors ◽  
Synthetic Biology ◽  
Directed Evolution ◽  
Cellular Systems ◽  
Intracellular Metabolite ◽  
Genetic Circuits ◽  
Synthetic Promoters ◽  
Genetic Switches ◽  
Metabolite Concentrations ◽  
Yeast Genetic

Genetic switches can be utilized for many purposes in synthetic biology including the assembly of complex genetic circuits to achieve sophisticated cellular systems and the construction of biosensors for real-time monitoring of intracellular metabolite concentrations. Although genetic switches have mainly been developed in prokaryotes to date, eukaryotic genetic switches are increasingly being reported as both rational and irrational engineering technologies mature. In this review, we describe genetic switches in yeast based on synthetic transcription factors and/or synthetic promoters. We also discuss directed evolution technologies for the rapid and robust construction of yeast genetic switches.

scholarly journals Saccharomyces cerevisiae Promoter Engineering before and during the Synthetic Biology Era

Biology ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 504
Author(s):  
Xiaofan Feng ◽  
Mario Andrea Marchisio
Keyword(s):  
Synthetic Biology ◽  
Dna Sequences ◽  
Genetic Circuits ◽  
Gene Circuits ◽  
Promoter Engineering ◽  
Rna Molecules ◽  
Promoter Sequences ◽  
Synthetic Promoters ◽  
Synthetic Gene Circuits ◽  
Wet Lab

Synthetic gene circuits are made of DNA sequences, referred to as transcription units, that communicate by exchanging proteins or RNA molecules. Proteins are, mostly, transcription factors that bind promoter sequences to modulate the expression of other molecules. Promoters are, therefore, key components in genetic circuits. In this review, we focus our attention on the construction of artificial promoters for the yeast S. cerevisiae, a popular chassis for gene circuits. We describe the initial techniques and achievements in promoter engineering that predated the start of the Synthetic Biology epoch of about 20 years. We present the main applications of synthetic promoters built via different methods and discuss the latest innovations in the wet-lab engineering of novel promoter sequences.

Biomolecular Computing Devices in Synthetic Biology

2010 ◽  
Vol 2 (2) ◽  
pp. 47-64
Author(s):  
Jesús M. Miró ◽  
Alfonso Rodríguez-Patón
Keyword(s):  
Synthetic Biology ◽  
Gene Expression Regulation ◽  
Logic Gates ◽  
Restriction Enzymes ◽  
Regulatory Proteins ◽  
Biomolecular Computing ◽  
Gene Circuits ◽  
Building Information ◽  
Competitive Hybridization ◽  
Standard Molecular Biology

Synthetic biology and biomolecular computation are disciplines that fuse when it comes to designing and building information processing devices. In this chapter, we study several devices that are representative of this fusion. These are three gene circuits implementing logic gates, a DNA nanodevice and a biomolecular automaton. The operation of these devices is based on gene expression regulation, the so-called competitive hybridization and the workings of certain biomolecules like restriction enzymes or regulatory proteins. Synthetic biology, biomolecular computation, systems biology and standard molecular biology concepts are also defined to give a better understanding of the chapter. The aim is to acquaint readers with these biomolecular devices born of the marriage between synthetic biology and biomolecular computation.

scholarly journals Phage-Based Applications in Synthetic Biology

2018 ◽  
Vol 5 (1) ◽  
pp. 453-476 ◽  
Author(s):  
Sebastien Lemire ◽  
Kevin M. Yehl ◽  
Timothy K. Lu
Keyword(s):  
Synthetic Biology ◽  
Molecular Biology ◽  
Directed Evolution ◽  
Antibacterial Agents ◽  
Biological Systems ◽  
Biological Functions ◽  
Next Generation ◽  
Gene Circuits ◽  
Tunable Properties

Bacteriophage research has been instrumental to advancing many fields of biology, such as genetics, molecular biology, and synthetic biology. Many phage-derived technologies have been adapted for building gene circuits to program biological systems. Phages also exhibit significant medical potential as antibacterial agents and bacterial diagnostics due to their extreme specificity for their host, and our growing ability to engineer them further enhances this potential. Phages have also been used as scaffolds for genetically programmable biomaterials that have highly tunable properties. Furthermore, phages are central to powerful directed evolution platforms, which are being leveraged to enhance existing biological functions and even produce new ones. In this review, we discuss recent examples of how phage research is influencing these next-generation biotechnologies.

scholarly journals Mammalian synthetic biology: emerging medical applications

2015 ◽  
Vol 12 (106) ◽  
pp. 20141000 ◽  
Author(s):  
Zoltán Kis ◽  
Hugo Sant'Ana Pereira ◽  
Takayuki Homma ◽  
Ryan M. Pedrigi ◽  
Rob Krams
Keyword(s):  
Drug Delivery ◽  
Synthetic Biology ◽  
Gene Networks ◽  
Regulatory Networks ◽  
Logic Gates ◽  
Boolean Logic ◽  
Synthetic Gene ◽  
Medical Applications ◽  
Synthetic Gene Networks ◽  
Gene Circuits

In this review, we discuss new emerging medical applications of the rapidly evolving field of mammalian synthetic biology. We start with simple mammalian synthetic biological components and move towards more complex and therapy-oriented gene circuits. A comprehensive list of ON–OFF switches, categorized into transcriptional, post-transcriptional, translational and post-translational, is presented in the first sections. Subsequently, Boolean logic gates, synthetic mammalian oscillators and toggle switches will be described. Several synthetic gene networks are further reviewed in the medical applications section, including cancer therapy gene circuits, immuno-regulatory networks, among others. The final sections focus on the applicability of synthetic gene networks to drug discovery, drug delivery, receptor-activating gene circuits and mammalian biomanufacturing processes.

scholarly journals Synthetic biology for the directed evolution of protein biocatalysts: navigating sequence space intelligently

2015 ◽  
Vol 44 (5) ◽  
pp. 1172-1239 ◽  
Author(s):  
Andrew Currin ◽  
Neil Swainston ◽  
Philip J. Day ◽  
Douglas B. Kell
Keyword(s):  
Synthetic Biology ◽  
Directed Evolution ◽  
Sequence Space ◽  
Search Spaces

Improving enzymes by directed evolution requires the navigation of very large search spaces; we survey how to do this intelligently.

Directed Evolution of The PobR Allosteric Transcription Factor To Generate A Biosensor For 4-Hydroxymandelic Acid

2021 ◽  
Author(s):  
YaoYao Liang ◽  
Juan Luo ◽  
Chenhao Yang ◽  
Shuning Guo ◽  
Bowen Zhang ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcription Factors ◽  
Directed Evolution ◽  
Missense Mutation ◽  
Hydroxybenzoic Acid ◽  
Ligand Specificity ◽  
Mutant Library

Abstract 4-Hydroxymandelic acid (HMA) is widely applied in pharmaceuticals, food and cosmetics. In this study, we aimed to develop an allosteric transcription factors (aTFs) based biosensor for HMA. PobR, an aTF for HMA analog 4-hydroxybenzoic acid, was used to alter its selectivity and create novel aTFs responsive to HMA by directed evolution. We established a PobR mutant library with a capacity of 550,000 mutants using error-prone PCR and Megawhop PCR. Through our screening, two mutants were obtained with responsiveness to HMA. Analysis of each missense mutation indicating residues 122-126 were involved in its PobR ligand specificity. These results showed the effectiveness of directed evolution in switching the ligand specificity of a biosensor and improving HMA production.

scholarly journals Blueprints for Biosensors: Design, Limitations, and Applications

Genes ◽  
2018 ◽  
Vol 9 (8) ◽  
pp. 375 ◽  
Author(s):  
Alexander Carpenter ◽  
Ian Paulsen ◽  
Thomas Williams
Keyword(s):  
Transcription Factors ◽  
Synthetic Biology ◽  
Nucleic Acids ◽  
Dynamic Range ◽  
Optimal Performance ◽  
Ease Of Use ◽  
Ligand Specificity ◽  
Biotechnological Application ◽  
Wide Range ◽  
Output Time

Biosensors are enabling major advances in the field of analytics that are both facilitating and being facilitated by advances in synthetic biology. The ability of biosensors to rapidly and specifically detect a wide range of molecules makes them highly relevant to a range of industrial, medical, ecological, and scientific applications. Approaches to biosensor design are as diverse as their applications, with major biosensor classes including nucleic acids, proteins, and transcription factors. Each of these biosensor types has advantages and limitations based on the intended application, and the parameters that are required for optimal performance. Specifically, the choice of biosensor design must consider factors such as the ligand specificity, sensitivity, dynamic range, functional range, mode of output, time of activation, ease of use, and ease of engineering. This review discusses the rationale for designing the major classes of biosensor in the context of their limitations and assesses their suitability to different areas of biotechnological application.

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