Synthetic Biology: A New Tool for the Trade.

Protein-protein interactions are fundamental to many biological processes. Yet, the weak and transient noncovalent bonds that characterize most protein-protein interactions found in nature impose limits on many bioengineering experiments. Here, a new class of genetically encodable peptide-protein pairs--isopeptag-N/pilin-N, isopeptag/pilin-C, and SpyTag/SpyCatcher--that interact through autocatalytic intermolecular isopeptide bond formation is described. Reactions between peptide-protein pairs are specific, robust, orthogonal, and able to proceed under most biologically relevant conditions both in vitro and in vivo. As fusion constructs, they provide a handle on molecules of interest, both organic and inorganic, that can be grasped with an iron grip. Such stable interactions provide robust post-translational control over biological processes and open new opportunities in synthetic biology for engineering programmable and self-assembling protein nanoarchitectures.

Synthesis and patterning of tunable multiscale materials with engineered cells.

Many natural biological systems--such as biofilms, shells and skeletal tissues--are able to assemble multifunctional and environmentally responsive multiscale assemblies of living and non-living components. Here, by using inducible genetic circuits and cellular communication circuits to regulate Escherichia coli curli amyloid production, we show that E. coli cells can organize self-assembling amyloid fibrils across multiple length scales, producing amyloid-based materials that are either externally controllable or undergo autonomous patterning. We also interfaced curli fibrils with inorganic materials, such as gold nanoparticles (AuNPs) and quantum dots (QDs), and used these capabilities to create an environmentally responsive biofilm-based electrical switch, produce gold nanowires and nanorods, co-localize AuNPs with CdTe/CdS QDs to modulate QD fluorescence lifetimes, and nucleate the formation of fluorescent ZnS QDs. This work lays a foundation for synthesizing, patterning, and controlling functional composite materials with engineered cells.

Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.

Protein interactions with peptides generally have low thermodynamic and mechanical stability. Streptococcus pyogenes fibronectin-binding protein FbaB contains a domain with a spontaneous isopeptide bond between Lys and Asp. By splitting this domain and rational engineering of the fragments, we obtained a peptide (SpyTag) which formed an amide bond to its protein partner (SpyCatcher) in minutes. Reaction occurred in high yield simply upon mixing and amidst diverse conditions of pH, temperature, and buffer. SpyTag could be fused at either terminus or internally and reacted specifically at the mammalian cell surface. Peptide binding was not reversed by boiling or competing peptide. Single-molecule dynamic force spectroscopy showed that SpyTag did not separate from SpyCatcher until the force exceeded 1 nN, where covalent bonds snap. The robust reaction conditions and irreversible linkage of SpyTag shed light on spontaneous isopeptide bond formation and should provide a targetable lock in cells and a stable module for new protein architectures.

A Full-wave Solution of Deep Sources in the Lossy Human Head to Accurate Electroencephalography and Magnetoencephalography.

Introduction: Currents in the brain flow inside neurons and across their boundaries into the extracellular medium, create electric and magnetic fields. These fields, which contain suitable information on brain activity, can be measured by electroencephalography (EEG), magnetoencephalography (MEG), and direct neural imaging. Methods: In this paper, we employed an electromagnetic model of the neuron activity and human head to derive electric and magnetic fields (brain waves) using a full-wave approach (ie. without any approximation). Currently, the brain waves are only derived using the quasi-static approximation (QSA) of Maxwell's equations in electromagnetic theory. Results: As a result, source localization in brain imaging will produce some errors. So far, the error rate of the QSA on the output results of electric and magnetic fields has not been investigated. This issue has become more noticeable due to the increased sensitivity of modern electroencephalography (EEG) and magnetoencephalography (MEG) devices. This work introduces issues that QSA encounters in this problem and reveals the necessity of a full-wave solution. Then, a full-wave solution of the problem in closed-form format is presented for the first time. This solution is done in two scenarios: the source (active neurons) is in the center of a sphere, and when the source is out of the center but deeply inside the sphere. The first scenario is simpler, but the second one is much more complicated and is solved using a partial-wave series expression. Conclusion: One of the significant achievements of this model is improving the interpretation of EEG and MEG measurements, resulting in more accurate source localization.

Full-colour Jabuticaba-like nanostructures via the multiplex and orthogonal self-assembly of protein-conjugated quantum dots with engineered biofilms.

The integration of inorganic components with bacterial biofilms is of great significance for expanding the functionality of artificial biological materials. However, so far, the complexities and functionalities of biofilm-based scaffolds assembled via metal-peptide coordination chemistries remain limited. Here, we present a platform for the multiplexed and specific coupling of recombinant protein-functionalized fluorescent red-green-blue (RGB) quantum dots (QDs) with engineered biofilms to form Jabuticaba-like nanostructures. Full-color living Jabuticaba-like nanostructures have been achieved through the interaction of extracellular peptides that are fabricated by biofilms with the proteins that modify the surface of the RGB QDs through orthogonal SpyTag/SpyCatcher, IsopeptagN/PilinN, and IsopeptagC/PilinC pairs. We envision that living cell populations will enable the multiplexable, scalable and bottom-up assembly of versatile materials that integrate both abiotic and biotic components into multifunctional systems.

Spontaneous intermolecular amide bond formation between side chains for irreversible peptide targeting.

Peptides and synthetic peptide-like molecules are powerful tools for analysis and control of biological function. One major limitation of peptides is the instability of their interactions with biomolecules, because of the limited accessible surface area for noncovalent interactions and the intrinsic flexibility of peptides. Peptide tags are nonetheless fundamental for protein detection and purification, because their small size minimizes the perturbation to protein function. Here we have designed a 16 amino acid peptide that spontaneously forms an amide bond to a protein partner, via reaction between lysine and asparagine side chains. This depended upon splitting a pilin subunit from a human pathogen, Streptococcus pyogenes, which usually undergoes intramolecular amide bond formation to impart mechanical and proteolytic stability to pili. Reaction of the protein partner was able to proceed to 98% conversion. The amide bond formation was independent of redox state and occurred at pH 5-8. The reaction was efficient in phosphate buffered saline and a wide range of biological buffers. Surprisingly, amide bond formation occurred at a similar rate at 4 and 37 degrees C. Both peptide and protein partners are composed of the regular 20 amino acids and reconstituted efficiently inside living E. coli. Labeling also showed high specificity on the surface of mammalian cells. Irreversible targeting of a peptide tag may have application in bioassembly, in cellular imaging, and to lock together proteins subject to high biological forces.

Multiplexed Sequence Encoding: A Framework for DNA Communication.

Synthetic DNA has great propensity for efficiently and stably storing non-biological information. With DNA writing and reading technologies rapidly advancing, new applications for synthetic DNA are emerging in data storage and communication. Traditionally, DNA communication has focused on the encoding and transfer of complete sets of information. Here, we explore the use of DNA for the communication of short messages that are fragmented across multiple distinct DNA molecules. We identified three pivotal points in a communication-data encoding, data transfer & data extraction-and developed novel tools to enable communication via molecules of DNA. To address data encoding, we designed DNA-based individualized keyboards (iKeys) to convert plaintext into DNA, while reducing the occurrence of DNA homopolymers to improve synthesis and sequencing processes. To address data transfer, we implemented a secret-sharing system-Multiplexed Sequence Encoding (MuSE)-that conceals messages between multiple distinct DNA molecules, requiring a combination key to reveal messages. To address data extraction, we achieved the first instance of chromatogram patterning through multiplexed sequencing, thereby enabling a new method for data extraction. We envision these approaches will enable more widespread communication of information via DNA.

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates.

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

DNA nanotechnology: new adventures for an old warhorse.

As the blueprint of life, the natural exploits of DNA are admirable. However, DNA should not only be viewed within a biological context. It is an elegantly simple yet functionally complex chemical polymer with properties that make it an ideal platform for engineering new nanotechnologies. Rapidly advancing synthesis and sequencing technologies are enabling novel unnatural applications for DNA beyond the realm of genetics. Here we explore the chemical biology of DNA nanotechnology for emerging applications in communication and digital data storage. Early studies of DNA as an alternative to magnetic and optical storage mediums have not only been promising, but have demonstrated the potential of DNA to revolutionize the way we interact with digital data in the future.

Superglue from bacteria: unbreakable bridges for protein nanotechnology.

Biotechnology is often limited by weak interactions. We suggest that an ideal interaction between proteins would be covalent, specific, require addition of only a peptide tag to the protein of interest, and form under a wide range of conditions. Here we summarize peptide tags that are able to form spontaneous amide bonds, based on harnessing reactions of adhesion proteins from the bacterium Streptococcus pyogenes. These include the irreversible peptide-protein interaction of SpyTag with SpyCatcher, as well as irreversible peptide-peptide interactions via SpyLigase. We describe existing applications, including polymerization to enhance cancer cell capture, assembly of living biomaterial, access to diverse protein shapes, and improved enzyme resilience. We also indicate future opportunities for resisting biological force and extending the scope of protein nanotechnology.

Synthetic biology of antimicrobial discovery.

Antibiotic discovery has a storied history. From the discovery of penicillin by Sir Alexander Fleming to the relentless quest for antibiotics by Selman Waksman, the stories have become like folklore used to inspire future generations of scientists. However, recent discovery pipelines have run dry at a time when multidrug-resistant pathogens are on the rise. Nature has proven to be a valuable reservoir of antimicrobial agents, which are primarily produced by modularized biochemical pathways. Such modularization is well suited to remodeling by an interdisciplinary approach that spans science and engineering. Herein, we discuss the biological engineering of small molecules, peptides, and non-traditional antimicrobials and provide an overview of the growing applicability of synthetic biology to antimicrobials discovery.

Structure and function of the glycopeptide N-methyltransferase MtfA, a tool for the biosynthesis of modified glycopeptide antibiotics.

There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the Amycolatopsis orientalis (S)-adenosyl-L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (S)-adenosyl-L-methionine, (S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methyl transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs.

Chemical biology of tetracycline antibiotics.

For more than half a century, tetracycline antibiotics have been used to treat infectious disease. However, what once used to be a commonly prescribed family of antibiotics has now decreased in effectiveness due to wide-spread bacterial resistance. The chemical scaffold of the tetracyclines is a versatile and modifiable structure that is able to interact with many cellular targets. The recent availability of detailed molecular interactions between tetracycline and its cellular targets, along with an understanding of the tetracycline biosynthetic pathway, has provided us with a unique opportunity to usher in a new era of rational drug design. Herein we discuss recent findings that have clarified the mode of action and the biosynthetic pathway of tetracyclines and that have shed light on the chemical biology of tetracycline antibiotics.