Modification of 3D-Printed PLA Structures Using Photo-Iniferter Polymerization: Toward On-Demand Antimicrobial Water Filters.

Water filtration is an important application to ensure the accessibility of clean drinking water. As requirements and contaminants vary on a local level, adjustable filter devices and their evaluation with contaminants are required. Within this work, modular filter devices are designed featuring an adjustable surface functionalization. For this purpose, 3D-printed structures are created consisting of bio-based poly(lactic acid) (PLA) that are manufactured by extrusion printing. The surface of PLA is activated with amino groups that are used to install xanthates as chain transfer agents. Subsequently, photo-iniferter (PI) polymerization is used to create cationic polymer brushes on the surface of PLA substrates. Multiple surface characterization techniques are employed to prove successful growth of polymer brushes on PLA. After initial optimization studies on flat surfaces, filter devices are printed, functionalized, and used to remove bacteria from contaminated water. Significant reduction of the number of microorganisms is detected after filtration (single filtration or cycling) and contaminating organism can also be removed from freshwater samples by simple incubation with a 3D-printed filter. The herein developed setup for producing functional filter devices and probing their performance in affinity filtration is a useful platform technology, enabling the rapid testing of polymer brushes for such applications.

Detection of Incorporation of p-Coumaric Acid into Photoactive Yellow Protein Variants in Vivo.

We report the design and characterization of photoactive yellow protein (PYP)-blue fluorescent protein (mTagBFP) fusion constructs that permit the direct assay of reconstitution and function of the PYP domain. These constructs allow for in vivo testing of co-expression systems for enzymatic production of the p-coumaric acid-based PYP chromophore, via the action of tyrosine ammonia lyase and p-coumaroyl-CoA ligase (pCL or 4CL). We find that different 4CL enzymes can function to reconstitute PYP, including 4CL from Arabidopsis thaliana that can produce ∼100% holo-PYP protein under optimal conditions. mTagBFP fusion constructs additionally enable rapid analysis of effects of mutations on PYP photocycles. We use this mTagBFP fusion strategy to demonstrate in vivo reconstitution of several PYP-based optogenetic tools in Escherichia coli via a biosynthesized chromophore, an important step for the use of these optogenetic tools in vivo in diverse hosts.

rAAV Engineering for Capsid-Protein Enzyme Insertions and Mosaicism Reveals Resilience to Mutational, Structural and Thermal Perturbations.

Recombinant adeno-associated viruses (rAAV) provide outstanding options for customization and superior capabilities for gene therapy. To access their full potential, facile genetic manipulation is pivotal, including capsid loop modifications. Therefore, we assessed capsid tolerance to modifications of the structural VP proteins in terms of stability and plasticity. Flexible glycine-serine linkers of increasing sizes were, at the genetic level, introduced into the 587 loop region of the VP proteins of serotype 2, the best studied AAV representative. Analyses of biological function and thermal stability with respect to genome release of viral particles revealed structural plasticity. In addition, insertion of the 29 kDa enzyme ß-lactamase into the loop region was tested with a complete or a mosaic modification setting. For the mosaic approach, investigation of VP2 trans expression revealed that a Kozak sequence was required to prevent leaky scanning. Surprisingly, even the full capsid modification with ß-lactamase allowed for the assembly of capsids with a concomitant increase in size. Enzyme activity assays revealed lactamase functionality for both rAAV variants, which demonstrates the structural robustness of this platform technology.

Long-range transcriptional interference in E. coli used to construct a dual positive selection system for genetic switches.

We have investigated transcriptional interference between convergent genes in E. coli and demonstrate substantial interference for inter-promoter distances of as far as 3 kb. Interference can be elicited by both strong σ(70) dependent and T7 promoters. In the presented design, a strong promoter driving gene expression of a 'forward' gene interferes with the expression of a 'reverse' gene by a weak promoter. This arrangement allows inversely correlated gene expression without requiring further regulatory components. Thus, modulation of the activity of the strong promoter alters expression of both the forward and the reverse gene. We used this design to develop a dual selection system for conditional operator site binding, allowing positive selection both for binding and for non-binding to DNA. This study demonstrates the utility of this novel system using the Lac repressor as a model protein for conditional DNA binding, and spectinomycin and chloramphenicol resistance genes as positive selection markers in liquid culture. Randomized LacI libraries were created and subjected to subsequent dual selection, but mispairing IPTG and selection cues in respect to the wild-type LacI response, allowing the isolation of a LacI variant with a reversed IPTG response within three rounds of library generation and dual selection.

Improving coiled coil stability while maintaining specificity by a bacterial hitchhiker selection system.

The design and selection of peptides targeting cellular proteins is challenging and often yields candidates with undesired properties. Therefore we deployed a new selection system based on the twin-arginine translocase (TAT) pathway of Escherichia coli, named hitchhiker translocation (HiT) selection. A pool of α-helix encoding sequences was designed and selected for interference with the coiled coil domain (CC) of a melanoma-associated basic-helix-loop-helix-leucine-zipper (bHLHLZ) protein, the microphthalmia associated transcription factor (MITF). One predominant sequence (iM10) was enriched during selection and showed remarkable protease resistance, high solubility and thermal stability while maintaining its specificity. Furthermore, it exhibited nanomolar range affinity towards the target peptide. A mutation screen indicated that target-binding helices of increased homodimer stability and improved expression rates were preferred in the selection process. The crystal structure of the iM10/MITF-CC heterodimer (2.1Å) provided important structural insights and validated our design predictions. Importantly, iM10 did not only bind to the MITF coiled coil, but also to the markedly more stable HLHLZ domain of MITF. Characterizing the selected variants of the semi-rational library demonstrated the potential of the innovative bacterial selection approach.

Analysis of selected and designed chimeric D- and L-α-helix assemblies.

D-peptides have been attributed pharmacological advantages over regular L-peptides, yet design rules are largely unknown. Based on a designed coiled coil-like D/L heterotetramer, named L-Base/D-Acid, we generated a library offering alternative residues for interaction with the D-peptide. Phage display selection yielded one predominant peptide, named HelixA, that differed at 13 positions from the scaffold helix. In addition to the observed D-/L-heterotetramers, ratio-dependent intermediate states were detected by isothermal titration calorimetry. Importantly, the formation of the selected HelixA/D-Acid bundle passes through fewer intermediate states than L-Base/D-Acid. Back mutation of HelixA core residues to L-Base (HelixLL) revealed that the residues at e/g-positions are responsible for the different intermediates. Furthermore, a Val-core variant (PeptideVV) was completely devoid of binding D-Acid, whereas an Ile-core helix (HelixII) interacted with D-Acid in a significantly more specific complex than L-Base.

Characterization and inhibition of AF10-mediated interaction.

The non-random chromosomal translocations t(10;11)(p13;q23) and t(10;11)(p13;q14-21) result in leukemogenic fusion proteins comprising the coiled coil domain of the transcription factor AF10 and the proteins MLL or CALM, respectively, and subsequently cause certain types of acute leukemia. The AF10 coiled-coil domain, which is crucial for the leukemogenic effect, has been shown to interact with GAS41, a protein previously identified as the product of an amplified gene in glioblastoma. Using sequential synthetic peptides, we mapped the potential AF10/GAS41 interaction site, which was subsequently be used as scaffold for a library targeting the AF10 coiled-coil domain. Using phage display, we selected a peptide that binds the AF10 coiled-coil domain with higher affinity than the respective coiled-coil region of wild-type GAS41, as demonstrated by phage ELISA, CD, and PCAs. Furthermore, we were able to successfully deploy the inhibitory peptide in a mammalian cell line to lower the expression of Hoxa genes that have been described to be overexpressed in these leukemias. This work dissects molecular determinants mediating AF10-directed interactions in leukemic fusions comprising the N-terminal parts of the proteins MLL or CALM and the C-terminal coiled-coil domain of AF10. Furthermore, it outlines the first steps in recognizing and blocking the leukemia-associated AF10 interaction in histiocytic lymphoma cells and therefore, may have significant implications in future diagnostics and therapeutics.

Directional cloning of DNA fragments using deoxyinosine-containing oligonucleotides and endonuclease V.

BACKGROUND: DNA fragments carrying internal recognition sites for the restriction endonucleases intended for cloning into a target plasmid pose a challenge for conventional cloning. RESULTS: A method for directional insertion of DNA fragments into plasmid vectors has been developed. The target sequence is amplified from a template DNA sample by PCR using two oligonucleotides each containing a single deoxyinosine base at the third position from the 5' end. Treatment of such PCR products with endonuclease V generates 3' protruding ends suitable for ligation with vector fragments created by conventional restriction endonuclease reactions. CONCLUSIONS: The developed approach generates terminal cohesive ends without the use of Type II restriction endonucleases, and is thus independent from the DNA sequence. Due to PCR amplification, minimal amounts of template DNA are required. Using the robust Taq enzyme or a proofreading Pfu DNA polymerase mutant, the method is applicable to a broad range of insert sequences. Appropriate primer design enables direct incorporation of terminal DNA sequence modifications such as tag addition, insertions, deletions and mutations into the cloning strategy. Further, the restriction sites of the target plasmid can be either retained or removed.

Exploring the molecular linkage of protein stability traits for enzyme optimization by iterative truncation and evolution.

The stability of proteins is paramount for their therapeutic and industrial use and, thus, is a major task for protein engineering. Several types of chemical and physical stabilities are desired, and discussion revolves around whether each stability trait needs to be addressed separately and how specific and compatible stabilizing mutations act. We demonstrate a stepwise perturbation-compensation strategy, which identifies mutations rescuing the activity of a truncated TEM ß-lactamase. Analyses relating structural stress with the external stresses of heat, denaturants, and proteases reveal our second-site suppressors as general stability centers that also improve the full-length enzyme. A library of lactamase variants truncated by 15 N-terminal and three C-terminal residues (Bla-NΔ15CΔ3) was subjected to activity selection and DNA shuffling. The resulting clone with the best in vivo performance harbored eight mutations, surpassed the full-length wild-type protein by 5.3 °C in T(m), displayed significantly higher catalytic activity at elevated temperatures, and showed delayed guanidine-induced denaturation. The crystal structure of this mutant was determined and provided insights into its stability determinants. Stepwise reconstitution of the N- and C-termini increased its thermal, denaturant, and proteolytic resistance successively, leading to a full-length enzyme with a T(m) increased by 15.3 °C and a half-denaturation concentration shifted from 0.53 to 1.75 M guanidinium relative to that of the wild type. These improvements demonstrate that iterative truncation-optimization cycles can exploit stability-trait linkages in proteins and are exceptionally suited for the creation of progressively stabilized variants and/or downsized proteins without the need for detailed structural or mechanistic information.

Strategies for Enzymatic Inactivation of the Veterinary Antibiotic Florfenicol.

Large quantities of the antibiotic florfenicol are used in animal farming and aquaculture, contaminating the ecosystem with antibiotic residues and promoting antimicrobial resistance, ultimately leading to untreatable multidrug-resistant pathogens. Florfenicol-resistant bacteria often activate export mechanisms that result in resistance to various structurally unrelated antibiotics. We devised novel strategies for the enzymatic inactivation of florfenicol in different media, such as saltwater or milk. Using a combinatorial approach and selection, we optimized a hydrolase (EstDL136) for florfenicol cleavage. Reaction kinetics were followed by time-resolved NMR spectroscopy. Importantly, the hydrolase remained active in different media, such as saltwater or cow milk. Various environmentally-friendly application strategies for florfenicol inactivation were developed using the optimized hydrolase. As a potential filter device for cost-effective treatment of waste milk or aquacultural wastewater, the hydrolase was immobilized on Ni-NTA agarose or silica as carrier materials. In two further application examples, the hydrolase was used as cell extract or encapsulated with a semi-permeable membrane. This facilitated, for example, florfenicol inactivation in whole milk, which can help to treat waste milk from medicated cows, to be fed to calves without the risk of inducing antibiotic resistance. Enzymatic inactivation of antibiotics, in general, enables therapeutic intervention without promoting antibiotic resistance.

SynBioWave--a real-time communication platform for molecular and synthetic biology.

SUMMARY: Synthetic Biology is advanced by many users and relies on the assembly of genetic elements to devices, systems and finally genomes. SynBioWave is a software suite that enables multiple distributed users to analyze and construct genetic parts in real-time collaboration. It builds on Google Wave and provides an extensible robot-robot-user communication framework, a menu driven user interface, biological data handling including DAS and an internal database communication. We demonstrate its use by implementing robots for gene-data retrieval, manipulation and display. The initial development of SynBioWave demonstrates the power of the underlying Google Wave protocol for Synthetic Biology and lays the foundation for continuous and user-friendly extensions. Specialized wave-robots with a manageable set of capabilities will divide and conquer the complex task of creating a genome in silico. AVAILABILITY: The robot is available at SynBioWave@appspot.com, the source code at http://synbiowave.sourceforge.net

Role of hydrophobic and electrostatic interactions in coiled coil stability and specificity.

We have screened two coiled coil-forming libraries in which core a and electrostatic e/g positions have been partially randomized. We observed the relative ability of these residues to confer coiled coil stability using a protein-fragment complementation assay. Our studies continue with the Jun/Fos activator protein-1 (AP-1) leucine zipper complex, as it provides a valid therapeutic target, while representing one of the more simplistic examples of quaternary structure. In mammalian cells, 28 possible dimeric interactions result from combinations of cJun, JunB, JunD, cFos, FosB, Fra1, and Fra2. Consequently, peptides designed to target particular oncogenic members must bind with high affinity and also be specific if they are to function as desired. We have therefore tested the ability of core and electrostatic interactions to confer stable and specific peptides. A previously selected peptide (FosW) formed the template for the core and electrostatic libraries. The winner from the core randomization (FosW(Core)) bound specifically to cJun relative to cFos, FosB, Fra1, Fra2, and the FosW(Core) homodimer, as verified by thermal melting analyses and growth competitions in the presence of either a negative control "mock" peptide or a competitor fusion peptide (cFos-FosB-Fra1-Fra2). In contrast, the winner from the electrostatic e/g randomization (FosW(e/g)) bound to all respective complexes with high stability, suggesting that the more significant energetic contributions made by core residues may be enough to generate specificity as a consequence of positive design.

Selection of peptides interfering with protein-protein interaction.

Cell physiology depends on a fine-tuned network of protein-protein interactions, and misguided interactions are often associated with various diseases. Consequently, peptides, which are able to specifically interfere with such adventitious interactions, are of high interest for analytical as well as medical purposes. One of the most abundant protein interaction domains is the coiled-coil motif, and thus provides a premier target. Coiled coils, which consist of two or more alpha-helices wrapped around each other, have one of the simplest interaction interfaces, yet they are able to confer highly specific homo- and heterotypic interactions involved in virtually any cellular process. While there are several ways to generate interfering peptides, the combination of library design with a powerful selection system seems to be one of the most effective and promising approaches. This chapter guides through all steps of such a process, starting with library options and cloning, detailing suitable selection techniques and ending with purification for further down-stream characterization. Such generated peptides will function as versatile tools to interfere with the natural function of their targets thereby illuminating their down-stream signaling and, in general, promoting understanding of factors leading to specificity and stability in protein-protein interactions. Furthermore, peptides interfering with medically relevant proteins might become important diagnostics and therapeutics.

Selection of Protein-Protein Interactions of Desired Affinities with a Bandpass Circuit.

We have developed a genetic circuit in Escherichia coli that can be used to select for protein-protein interactions of different strengths by changing antibiotic concentrations in the media. The genetic circuit links protein-protein interaction strength to ß-lactamase activity while simultaneously imposing tuneable positive and negative selection pressure for ß-lactamase activity. Cells only survive if they express interacting proteins with affinities that fall within set high- and low-pass thresholds; i.e. the circuit therefore acts as a bandpass filter for protein-protein interactions. We show that the circuit can be used to recover protein-protein interactions of desired affinity from a mixed population with a range of affinities. The circuit can also be used to select for inhibitors of protein-protein interactions of defined strength.

Characterizing Transcriptional Interference between Converging Genes in Bacteria.

Antisense transcription is common in naturally occurring genomes and is increasingly being used in synthetic genetic circuitry as a tool for gene expression control. Mutual influence on the expression of convergent genes can be mediated by antisense RNA effects and by transcriptional interference (TI). We aimed to quantitatively characterize long-range TI between convergent genes with untranslated intergenic spacers of increasing length. After controlling for antisense RNA-mediated effects, which contributed about half of the observed total expression inhibition, the TI effect was modeled. To achieve model convergence, RNA polymerase processivity and collision resistance were assumed to be modulated by ribosome trailing. The spontaneous transcription termination rate in regions of untranslated DNA was experimentally determined. Our modeling suggests that an elongating RNA polymerase with a trailing ribosome is about 13 times more likely to resume transcription than an opposing RNA polymerase without a trailing ribosome, upon head-on collision of the two.

iPEP: peptides designed and selected for interfering with protein interaction and function.

Semi-rational design is combined with PCAs (protein-fragment complementation assays) and phage-display screening techniques to generate a range of iPEPs (interfering peptides) that target therapeutically relevant proteins with much higher interaction stability than their native complexes. PCA selection has been improved to impose a competitive and negative design initiative on the library screen, thus simultaneously improving the specificity of assay 'winners'. The folding pathways of designed pairs imply that early events are dominated by hydrophobic collapse and helix formation, whereas later events account for the consolidation of more intricate intermolecular electrostatic interactions.

Considerations in the design and optimization of coiled coil structures.

Coiled coil motifs are, despite their apparent simplicity, highly specific, and play a significant role in the understanding of tertiary structure and its formation. The most commonly observed of the coiled coils, the parallel dimeric, is yet to be fully characterized for this structural class in general. Nonetheless, strict rules have emerged for the necessity of specific types of amino acids at specific positions. In this chapter, we discuss this system in light of existing coiled coil structures and in applying rules to coiled coils that are to be designed or optimized. Understanding and expanding on these rules is crucial in using these motifs, which play key roles in virtually every cellular process, to act as drug-delivery agents by sequestering other proteins that are not behaving natively or that have been upregulated (for example, by binding to coiled coil domains implicated in oncogenesis). The roles of the a and d "hydrophobic" core positions and the e and g "electrostatic" edge positions in directing oligomerization and pairing specificity are discussed. Also discussed is the role of these positions in concert with the b, c, and f positions in maintaining alpha-helical propensity, helix solubility, and dimer stability.

Versatile DNA fragmentation and directed evolution with nucleotide exchange and excision technology.

Mimicking natural evolution by DNA shuffling is a commonly used method for the optimization of DNA and protein properties. Here, we present an advancement of this approach whereby a gene library is amplified using a standard polymerase chain reaction (PCR), but incorporates dUTP as a fragmentation-defining exchange nucleotide, together with the four standard dNTPs. Incorporated uracil bases are excised using uracil-DNA-glycosylase, and the DNA backbone subsequently is cleaved with piperidine. This oligonucleotide pool is then reassembled with an internal primer extension procedure using a proofreading polymerase to increase yield, and, finally, is amplified by PCR. Denaturing polyacrylamide urea gels demonstrate this method to produce adjustable fragmentation size ranges dependent on the dUTP:dTTP ratios. Using the model protein, chloramphenicol acetyltransferase I, the sequencing of shuffled gene libraries based on a PCR containing 33% dUTP revealed a low mutation rate, of approx 0.1%, with an average parental fragments size of 86 bases, even without the use of a fragment-size separation. Nucleotide exchange and excision technology (NExT) DNA shuffling is, thus, reproducible and easily executed, making it superior to competing techniques. Additionally, NExT fragmentation outcome can be predicted using the computer software, NExTProg.

A general method of terminal truncation, evolution, and re-elongation to generate enzymes of enhanced stability.

Improving enzyme stability is a highly desirable design step in generating enzymes able to function under extreme conditions, such as elevated temperatures, while having the additional benefit of being less susceptible to cleavage by proteases. For these reasons, many different approaches and techniques have been devised in constructing such proteins, but the results to date have been of mixed success. Here, we present a robust method involving the terminal truncation, random mutagenesis and fragmentation, recombination, elongation, and finally, selection at physiological temperatures, to generate an enzyme with improved stability. Three cycles of directed evolution comprising of random mutagenesis, DNA shuffling, and selection at 37 degrees C were used, using the bacterial enzyme TEM-1 beta-lactamase as a model protein to yield deletion mutants with in vivo ampicillin resistance levels comparable to wild-type (wt) enzyme. Kinetic studies demonstrate the selected mutant to have a significantly improved thermostability relative to its wt counterpart. Elongation of this mutant to the full-length gene resulted in a beta-lactamase variant with dramatically increased thermostability. This technique was so fruitful that the evolved enzyme retained its maximum catalytic activity even 20 degrees C above its wt parent protein optimum. Thus, structural perturbation by terminal truncation and subsequent compensation by directed evolution at physiological temperatures is a fast, efficient, and highly effective way to improve the thermostability of proteins without the need for selecting at elevated temperatures.

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution.

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.