Laboratory automation with robot-assisted processes enhances synthetic biology, but its economic impact on projects is uncertain. We have proposed an experiment price index (EPI) for a quantitative ...comparison of factors in time, cost, and sample numbers, helping measure the efficiency of laboratory automation in synthetic biology and biomolecular engineering.
Laboratory automation with robot-assisted processes enhances synthetic biology, but its economic impact on projects is uncertain. We have proposed an experiment price index (EPI) for a quantitative comparison of factors in time, cost, and sample numbers, helping measure the efficiency of laboratory automation in synthetic biology and biomolecular engineering.
Display omitted
•We review affinity assays that are based on aptamer binding to thrombin.•Thrombin is most frequently used to demonstrate the proof-of-principle.•The principles for thrombin assays ...are applicable to other molecular targets.•The thrombin example describes key features of aptamer affinity assays.
Experimentally selected single-stranded DNA and RNA aptamers are able to bind to specific target molecules with high affinity and specificity. Many analytical methods make use of affinity binding between the specific targets and their aptamers. In the development of these methods, thrombin is the most frequently used target molecule to demonstrate the proof-of-principle. This paper critically reviews more than one hundred assays that are based on aptamer binding to thrombin. This review focuses on homogeneous binding assays, electrochemical aptasensors, and affinity separation techniques. The emphasis of this review is placed on understanding the principles and unique features of the assays. The principles of most assays for thrombin are applicable to the determination of other molecular targets.
Synthetic Biology is a rapidly growing interdisciplinary field that is primarily built upon foundational advances in molecular biology combined with engineering design principles such as modularity ...and interoperability. The field considers living systems as programmable at the genetic level and has been defined by the development of new platform technologies and methodological advances. A key concept driving the field is the Design-Build-Test-Learn cycle which provides a systematic framework for building new biological systems. One major application area for synthetic biology is biosynthetic pathway engineering that requires the modular assembly of different genetic regulatory elements and biosynthetic enzymes. In this review we provide an overview of modular DNA assembly and describe and compare the plethora of in vitro and in vivo assembly methods for combinatorial pathway engineering. Considerations for part design and methods for enzyme balancing are also presented, and we briefly discuss alternatives to intracellular pathway assembly including microbial consortia and cell-free systems for biosynthesis. Finally, we describe computational tools and automation for pathway design and assembly and argue that a deeper understanding of the many different variables of genetic design, pathway regulation and cellular metabolism will allow more predictive pathway design and engineering.
•Coverage of multipart DNA assembly methods and organism-specific part collections.•Considerations for the design of genetic elements required for a metabolic pathway.•Enzyme balancing toolkits that do not require a pathway-specific DNA library.•Analysis of current computational tools and automation methods to assist assembly.
Metallic bowtie nanoarchitectures can produce dramatic electric field enhancement, which is advantageous in single‐molecule analysis and optical information processing. Plasmonic bowtie ...nanostructures were successfully constructed using a DNA origami‐based bottom‐up assembly strategy, which enables precise control over the geometrical configuration of the bowtie with an approximate 5 nm gap. A single Raman probe was accurately positioned at the gap of the bowtie. Single‐molecule surface‐enhanced Raman scattering (SM‐SERS) of individual nanostructures, including ones containing an alkyne group, was observed. The design achieved repeatable local field enhancement of several orders of magnitude. This method opens the door on a novel strategy for the fabrication of metal bowtie structures and SM‐SERS, which can be utilized in the design of highly‐sensitive photonic devices.
Plasmonic bowtie nanostructures were successfully constructed using DNA origami‐based self‐assembly. A single Raman probe was accurately positioned at the gap of the bowtie and single‐molecule SERS of individual nanostructures was observed.
Class 2 CRISPR-Cas nucleases are programmable genome editing tools with promising applications in human health and disease. However, DNA cleavage at off-target sites that resemble the target sequence ...is a pervasive problem that remains poorly understood mechanistically. Here, we use quantitative kinetics to dissect the reaction steps of DNA targeting by Acidaminococcus sp Cas12a (also known as Cpf1). We show that Cas12a binds DNA tightly in two kinetically separable steps. Protospacer-adjacent motif (PAM) recognition is followed by rate-limiting R-loop propagation, leading to inevitable DNA cleavage of both strands. Despite functionally irreversible binding, Cas12a discriminates strongly against mismatches along most of the DNA target sequence. This result implies substantial reversibility during R-loop formation—a late transition state—and defies common descriptions of a “seed” region. Our results provide a quantitative basis for the DNA cleavage patterns measured in vivo and observations of greater reported target specificity for Cas12a than for the Cas9 nuclease.
Display omitted
•DNA target binding by CRISPR-Cas12a is rate limiting for DNA cleavage•Multiple cleavage sites are present on each DNA strand•Specificity against mismatches suggests a late transition state for R-loop formation•Data explain in vivo cleavage patterns by Cas12a and differences from Cas9
Strohkendl et al. dissect DNA binding and cleavage by CRISPR-Cas12a. They show that binding is functionally irreversible, yet Cas12a discriminates against mismatches with the target DNA extending beyond a seed region. These results suggest that R-loop propagation is readily reversible, enabling Cas12a to select DNA sequences more precisely than Cas9.
The bioengineering of nonribosomal peptide synthetases (NRPSs) is a rapidly developing field to access natural product derivatives and new-to-nature natural products like scaffolds with changed or ...improved properties. However, the rational (re-)design of these often gigantic assembly-line proteins is by no means trivial and needs in-depth insights into structural flexibility, inter-domain communication, and the role of proofreading by catalytic domains-so it is not surprising that most previous rational reprogramming efforts have been met with limited success. With this practical guide, the result of nearly one decade of NRPS engineering in the Bode lab, we provide valuable insights into the strategies we have developed during this time for the successful engineering and cloning of these fascinating molecular machines.
Synthetic biology relies on the manufacture of large and complex DNA constructs from libraries of genetic parts. Golden Gate and other Type IIS restriction enzyme-dependent DNA assembly methods ...enable rapid construction of genes and operons through one-pot, multifragment assembly, with the ordering of parts determined by the ligation of Watson–Crick base-paired overhangs. However, ligation of mismatched overhangs leads to erroneous assembly, and low-efficiency Watson Crick pairings can lead to truncated assemblies. Using sets of empirically vetted, high-accuracy junction pairs avoids this issue but limits the number of parts that can be joined in a single reaction. Here, we report the use of comprehensive end-joining ligation fidelity and bias data to predict high accuracy junction sets for Golden Gate assembly. The ligation profile accurately predicted junction fidelity in ten-fragment Golden Gate assembly reactions and enabled accurate and efficient assembly of a lac cassette from up to 24-fragments in a single reaction.
Competitive sustainable production in industry demands new and better biocatalysts, optimized bioprocesses and cost-effective product recovery. Our review sheds light on the progress made for the ...individual steps towards these goals, starting with the discovery of new enzymes and their corresponding genes. The enzymes are subsequently engineered to improve their performance, combined in reaction cascades to expand the reaction scope and integrated in whole cells to provide an optimal environment for the bioconversion. Strain engineering using synthetic biology methods tunes the host for production, reaction design optimizes the reaction conditions and downstream processing ensures the efficient recovery of commercially viable products. Selected examples illustrate how modified enzymes can revolutionize future-oriented applications ranging from the bioproduction of bulk-, specialty- and fine chemicals, active pharmaceutical ingredients and carbohydrates, over the conversion of the greenhouse-gas CO2 into valuable products and biocontrol in agriculture, to recycling of synthetic polymers and recovery of precious metals.
PDF
