We present a high throughput microfluidic device for continuous-flow polymerase chain reaction (PCR) in water-in-oil droplets of nanoliter volumes. The circular design of this device allows droplets to pass through alternating temperature zones and complete 34 cycles of PCR in only 17 min, avoiding temperature cycling of the entire device. The temperatures for the applied two-temperature PCR protocol can be adjusted according to requirements of template and primers. These temperatures were determined with fluorescence lifetime imaging (FLIM) inside the droplets, exploiting the temperature-dependent fluorescence lifetime of rhodamine B. The successful amplification of an 85 base-pair long template from four different start concentrations was demonstrated. Analysis of the product by gel-electrophoresis, sequencing, and real-time PCR showed that the amplification is specific and the amplification factors of up to 5 x 10(6)-fold are comparable to amplification factors obtained in a benchtop PCR machine. The high efficiency allows amplification from a single molecule of DNA per droplet. This device holds promise for convenient integration with other microfluidic devices and adds a critical missing component to the laboratory-on-a-chip toolkit.
A system is reported for the recombinant expression of individual ketoreductase (KR) domains from modular polyketide synthases (PKSs) and scrutiny of their intrinsic specificity and stereospecificity toward surrogate diketide substrates. The eryKR(1) and the tylKR(1) domains, derived from the first extension module of the erythromycin PKS and the tylosin PKS, respectively, both catalyzed reduction of (2R, S)-2-methyl-3-oxopentanoic acid N-acetylcysteamine thioester, with complete stereoselectivity and stereospecificity, even though the substrate is not tethered to an acyl carrier protein or an intact PKS multienzyme. In contrast, and to varying degrees, the isolated enzymes eryKR(2), eryKR(5), and eryKR(6) exercised poorer control over substrate selection and the stereochemical course of ketoreduction. These data, together with modeling of diketide binding to KR(1) and KR(2), demonstrate the fine energetic balance between alternative modes of presentation of ketoacylthioester substrates to KR active sites.
The nucleotide sequences of the infectious cloned DNA components of tomato golden mosaic virus (TGMV) have been determined. DNA A (2588 nucleotides) and DNA B (2508 nucleotides) have little sequence homology except for a region of ˜200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed six potential coding regions for proteins of mol. wt. >10 000, four in DNA A and two in DNA B. Possible regulatory signals are identified and a model for bidirectional transcription of the two genome components is presented. Comparison of the nucleotide sequences of the DNAs of TGMV and cassava latent virus (CLV) revealed a fairly close relationship between TGMV DNA A and CLV DNA 1 and a comparatively distant relationship between TGMV DNA B and CLV DNA 2. All the potential coding regions in the TGMV DNAs had counterparts in the CLV DNAs suggesting an overall similarity in genome organisation, but six potential coding regions in the CLV DNAs had no counterparts in the TGMV DNAs. The 200‐base region common to the two DNAs of each virus had little sequence homology, except for a highly conserved 33‐base sequence potentially capable of forming a stable hairpin structure.
The bottom-up design of protein-based signaling networks is a key goal of synthetic biology; yet, it remains elusive due to our inability to tailor-make signal transducers and receptors that can be readily compiled into defined signaling networks. Here, we report a generic approach for the construction of protein-based molecular switches based on artficially autoinhibited proteases. Using structure-guided design and directed protein evolution, we created signal transducers based on artificially autoinhibited proteases that can be activated following site-specific proteolysis and also demonstrate the modular design of an allosterically regulated protease receptor following recombination with an affinity clamp peptide receptor. Notably, the receptor's mode of action can be varied from >5-fold switch-OFF to >30-fold switch-ON solely by changing the length of the connecting linkers, demonstrating a high functional plasticity not previously observed in naturally occurring receptor systems. We also create an integrated signaling circuit based on two orthogonal autoinhibited protease units that can propagate and amplify molecular queues generated by the protease receptor. Finally, we present a generic two-component receptor architecture based on proximity-based activation of two autoinhibited proteases. Overall, the approach allows the design of protease-based signaling networks that, in principle, can be connected to any biological process.synthetic biology | protein engineering | protein switches | proteases A key objective in the emerging field of synthetic biology is to develop approaches for the systematic engineering of artificial signal transduction systems (1, 2), which is expected to advance our understanding of fundamental biological processes and create new biotechnological and medical applications (3). Engineering synthetic signaling systems have predominantly been realized with gene expression circuits that relay molecular queues either through transcription factors or functional nucleic acids (4-8). Here, rational engineering strategies are supported by the modular organization and function of transcription factors and regulatory DNA elements (4), as well as the predictability of base pairing interactions (8). However, transcription-based signaling circuits usually require hours to process and actuate a specific molecular queue (9). Further, the limited chemical diversity of nucleic acids compared with amino acids ultimately restricts their functionality. Thus, protein-based signaling networks that operate faster compared with gene-based signaling circuits are highly desirable.The systematic engineering of protein-based modules that sense, process, and amplify defined molecular queues has provided a formidable challenge to date. At the molecular level, many biological response functions depend on allosterically regulated protein activities that couple an input to an output function solely through conformational changes. Engineering these has proven difficult with only a few successful designs reported to ...
Biosensors with direct electron output hold promise for nearly seamless integration with portable electronic devices. However, so far, they have been based on naturally occurring enzymes that significantly limit the spectrum of detectable analytes. Here, we present a novel biosensor architecture based on analyte-driven intermolecular recombination and activity reconstitution of a re-engineered component of glucometers: PQQ-glucose dehydrogenase. We demonstrate that this sensor architecture can be rapidly adopted for the detection of immunosuppressant drugs, α-amylase protein, or protease activity of thrombin and Factor Xa. The biosensors could be stored in dried form without appreciable loss of activity. We further show that ligand-induced activity of the developed biosensors could be directly monitored by chronoamperometry, enabling construction of disposable sensory electrodes. We expect that this architecture could be expanded to the detection of other biochemical activities, post-translational modifications, nucleic acids, and inorganic molecules.
Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.
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