scholarly journals Standardizing Automated DNA Assembly: Best Practices, Metrics, and Protocols Using Robots

2019 ◽  
Vol 24 (3) ◽  
pp. 282-290 ◽  
Author(s):  
David I. Walsh ◽  
Marilene Pavan ◽  
Luis Ortiz ◽  
Scott Wick ◽  
Johanna Bobrow ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Best Practices ◽  
High Throughput ◽  
Dna Sequences ◽  
Biological Systems ◽  
Software Tool ◽  
Parameter Analysis ◽  
Dna Assembly ◽  
Assembly Design ◽  
Liquid Handling

The advancement of synthetic biology requires the ability to create new DNA sequences to produce unique behaviors in biological systems. Automation is increasingly employed to carry out well-established assembly methods of DNA fragments in a multiplexed, high-throughput fashion, allowing many different configurations to be tested simultaneously. However, metrics are required to determine when automation is warranted based on factors such as assembly methodology, protocol details, and number of samples. The goal of our synthetic biology automation work is to develop and test protocols, hardware, and software to investigate and optimize DNA assembly through quantifiable metrics. We performed a parameter analysis of DNA assembly to develop a standardized, highly efficient, and reproducible MoClo protocol, suitable to be used both manually and with liquid-handling robots. We created a key DNA assembly metric (Q-metric) to characterize a given automation method’s advantages over conventional manual manipulations with regard to researchers’ highest-priority parameters: output, cost, and time. A software tool called Puppeteer was developed to formally capture these metrics, help define the assembly design, and provide human and robotic liquid-handling instructions. Altogether, we contribute to a growing foundation of standardizing practices, metrics, and protocols for automating DNA assembly.

scholarly journals A set of experimentally validated, mutually orthogonal primers for combinatorially specifying genetic components

2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Subu K Subramanian ◽  
William P Russ ◽  
Rama Ranganathan
Keyword(s):  
Synthetic Biology ◽  
High Throughput ◽  
Dna Sequences ◽  
Gene Synthesis ◽  
Modular Architecture ◽  
Design And Synthesis ◽  
Genetic Components ◽  
Polymerase Chain ◽  
Polymerase Chain Reaction Primers ◽  
Microarray Chips

Abstract The design and synthesis of novel genes and deoxyribonucleic acid (DNA) sequences is a central technique in synthetic biology. Current methods of high throughput gene synthesis use pooled oligonucleotides obtained from custom-designed DNA microarray chips, and rely on orthogonal (non-interacting) polymerase chain reaction primers to specifically de-multiplex, by amplification, the precise subset of oligonucleotides necessary to assemble a full length gene. The availability of a large validated set of mutually orthogonal primers is therefore a crucial reagent for high-throughput gene synthesis. Here, we present a set of 166 20-nucleotide primers that are experimentally verified to be non-interacting, capable of specifying 13 695 unique genes. These primers represent a valuable resource to the synthetic biology community for specifying genetic components that can be assembled through a scalable and modular architecture.

scholarly journals The Five-Primer Challenge: An inquiry-based laboratory module for synthetic biology

2019 ◽  
Author(s):  
Suzie Hsu ◽  
Jay Huenemann ◽  
Vishwesh Kulkarni ◽  
Rodrigo Ledesma-Amaro ◽  
Robert Moseley ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequences ◽  
New Technologies ◽  
Dna Assembly ◽  
Gfp Expression ◽  
Student Centered ◽  
Bacterial Gene ◽  
Bacterial Gene Expression ◽  
Central Activity ◽  
Cold Spring

AbstractNew technologies in DNA synthesis and assembly give genetic engineers complete freedom in genetic design, where virtually any plasmid DNA sequence can be created efficiently and economically. Learning how to design, construct, and test new DNA sequences is a critical skill for researchers in molecular biology and biotechnology. Here we present a student-centered, inquiry-based module in which students learn how to control bacterial gene expression by appplying various DNA assembly techniques. The central activity in this learning module is termed the ‘Five-Primer Challenge’. Each student is allowed to order up to five 60-mer oligonucleotide primers to then modify a GFP expression plasmid with the goal of increasing GFP expression as much as possible. This module was developed and implemented at the 2016 Cold Spring Harbor Laboratory Synthetic Biology Course, and was effective at engaging students in critical thinking and in promoting student learning.

scholarly journals A fully automated pipeline for the dynamic at‐line morphology analysis of microscale Aspergillus cultivation

2021 ◽  
Vol 8 (1) ◽  
Author(s):  
Roman Jansen ◽  
Kira Küsters ◽  
Holger Morschett ◽  
Wolfgang Wiechert ◽  
Marco Oldiges
Keyword(s):  
Filamentous Fungi ◽  
High Throughput ◽  
Automated Image Analysis ◽  
Height Velocity ◽  
Pellet Size ◽  
Application Study ◽  
Liquid Handling ◽  
Morphology Analysis ◽  
Development And Utilization ◽  
Increasing Demand

Abstract Background Morphology, being one of the key factors influencing productivity of filamentous fungi, is of great interest during bioprocess development. With increasing demand of high-throughput phenotyping technologies for fungi due to the emergence of novel time-efficient genetic engineering technologies, workflows for automated liquid handling combined with high-throughput morphology analysis have to be developed. Results In this study, a protocol allowing for 48 parallel microbioreactor cultivations of Aspergillus carbonarius with non-invasive online signals of backscatter and dissolved oxygen was established. To handle the increased cultivation throughput, the utilized microbioreactor is integrated into a liquid handling platform. During cultivation of filamentous fungi, cell suspensions result in either viscous broths or form pellets with varying size throughout the process. Therefore, tailor-made liquid handling parameters such as aspiration/dispense height, velocity and mixing steps were optimized and validated. Development and utilization of a novel injection station enabled a workflow, where biomass samples are automatically transferred into a flow through chamber fixed under a light microscope. In combination with an automated image analysis concept, this enabled an automated morphology analysis pipeline. The workflow was tested in a first application study, where the projected biomass area was determined at two different cultivation temperatures and compared to the microbioreactor online signals. Conclusions A novel and robust workflow starting from microbioreactor cultivation, automated sample harvest and processing via liquid handling robots up to automated morphology analysis was developed. This protocol enables the determination of projected biomass areas for filamentous fungi in an automated and high-throughput manner. This measurement of morphology can be applied to describe overall pellet size distribution and heterogeneity.

scholarly journals A Synthetic Biology Approach Using Engineered Bacteria to Detect Perfluoroalkyl Substance (PFAS) Contamination in Water

2021 ◽  
Vol 186 (Supplement_1) ◽  
pp. 801-807
Author(s):  
Nathaniel A Young ◽  
Ryan L Lambert ◽  
Angela M Buch ◽  
Christen L Dahl ◽  
Jackson D Harris ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Dna Sequences ◽  
Detection System ◽  
The United States ◽  
Detection Methods ◽  
Health Concern ◽  
Contaminated Water ◽  
Engineered Bacteria ◽  
Synthetic Biology Approach ◽  
Rhodococcus Jostii

ABSTRACT Introduction Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds used industrially for a wide variety of applications. These PFAS compounds are very stable and persist in the environment. The PFAS contamination is a growing health issue as these compounds have been reported to impact human health and have been detected in both domestic and global water sources. Contaminated water found on military bases poses a potentially serious health concern for active duty military, their families, and the surrounding communities. Previous detection methods for PFAS in contaminated water samples require expensive and time-consuming testing protocols that limit the ability to detect this important global pollutant. The main objective of this work was to develop a novel detection system that utilizes a biological reporter and engineered bacteria as a way to rapidly and efficiently detect PFAS contamination. Materials and Methods The United States Air Force Academy International Genetically Engineered Machine team is genetically engineering Rhodococcus jostii strain RHA1 to contain novel DNA sequences composed of a propane 2-monooxygenase alpha (prmA) promoter and monomeric red fluorescent protein (mRFP). The prmA promoter is activated in the presence of PFAS and transcribes the mRFP reporter. Results The recombinant R. jostii containing the prmA promoter and mRFP reporter respond to exposure of PFAS by activating gene expression of the mRFP. At 100 µM of perfluorooctanoic acid, the mRFP expression was increased 3-fold (qRT-PCR). Rhodococcus jostii without exposure to PFAS compounds had no mRFP expression. Conclusions This novel detection system represents a synthetic biology approach to more efficiently detect PFAS in contaminated samples. With further refinement and modifications, a similar system could be readily deployed in the field around the world to detect this critical pollutant.

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

scholarly journals Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology

2017 ◽  
Vol 6 (11) ◽  
pp. 1988-1995 ◽  
Author(s):  
José M. Duarte ◽  
Içvara Barbier ◽  
Yolanda Schaerli
Keyword(s):  
Synthetic Biology ◽  
High Throughput ◽  
High Throughput Screening ◽  
Gel Beads

scholarly journals Erratum to: No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

2016 ◽  
Vol 10 (1) ◽  
Author(s):  
Afnan Azizi ◽  
Wilson Lam ◽  
Hilary Phenix ◽  
Lioudmila Tepliakova ◽  
Ian J. Roney ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Best Practice ◽  
Experimental Tests ◽  
Dna Assembly

scholarly journals Independent control of amplitude and period in a synthetic oscillator circuit with modified repressilator

2022 ◽  
Vol 5 (1) ◽  
Author(s):  
Fengyu Zhang ◽  
Yanhong Sun ◽  
Yihao Zhang ◽  
Wenting Shen ◽  
Shujing Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Biological Systems ◽  
Reporter Genes ◽  
Quantitative Model ◽  
Theoretical Level ◽  
Independent Control ◽  
Oscillator Circuit ◽  
Biological Circuits

AbstractSynthetic Biology aims to create predictable biological circuits and fully operational biological systems. Although there are methods to create more stable oscillators, such as repressilators, independently controlling the oscillation of reporter genes in terms of their amplitude and period is only on theoretical level. Here, we introduce a new oscillator circuit that can be independently controlled by two inducers in Escherichia coli. Some control components, including σECF11 and NahR, were added to the circuit. By systematically tuning the concentration of the inducers, salicylate and IPTG, the amplitude and period can be modulated independently. Furthermore, we constructed a quantitative model to forecast the regulation results. Under the guidance of the model, the expected oscillation can be regulated by choosing the proper concentration combinations of inducers. In summary, our work achieved independent control of the oscillator circuit, which allows the oscillator to be modularized and used in more complex circuit designs.

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