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Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated disease (PCVAD) that causes huge global economic losses for the swine industry. Effective strategies or rapid detection of PCV2 in pig are essential to control PCVAD. Here, single-chain variable fragments (scFvs) were selected and characterized against the PCV2 capsid using phage display technology. Phage scFv clones were selected from the human scFv phagemid library (Tomlinson I + J) for direct panning against the PCV2 capsid. Eighty-four monoclonal phage scFvs were individually tested for binding to the PCV2 capsid by ELISA. Eight scFv clones showed significant binding to the PCV2 capsid and only three clones (clone nos. 13, 37, and 81) contained both VHCDRs and VLCDRs in the sequence. Clone scFv no. 81 had the highest reactivity to the PCV2 capsid and was constructed in the pET22b (+) expression vector. The recombinant was transformed to Escherichia coli BL21(DE3) for expression and purification. The scFv showed appropriate affinity to the PCV2 capsid by western blot analysis. Kinetics of scFv and the PCV2 capsid were determined using surface plasmon resonance and showed binding affinity in the nanomolar range (K D = 57.2 nM). Our scFv was first applied in the development of an impedimetric immunosensor for PCV2 capsid detection, and results showed that impedance increased with increasing PCV2 capsid expression with limit of detection = 114 nM. Findings demonstrated that our scFv has potential for use as a receptor for biosensor devices.

DNA origami is a powerful tool for the production of self-assembled nanostructures and has shown promising applications in a wide range of fields. These structures are typically assembled through a reaction between one long single-stranded DNA (ssDNA) scaffold and many short oligonucleotide staples. In our efforts towards developing applications of DNA origami, we determined that two important properties for scaffold production include the ability to customize its size and sequence, and the ability to produce scaffolds cost effectively at scale. Several in vitro methods exist for producing custom scaffolds. However, because they rely on enzymatic processes, these methods are unreliable at large scale. On the other hand, scalable production of scaffolds based on the M13 bacteriophage genome has been demonstrated. However, the M13 genome will only accommodate insertions up to 2.5 kilobases (kb) reliably, limiting customizability. Likewise, phagemid vectors allow for the production of phage-based scaffolds with larger custom regions, but they still require 2.9 kb of fixed sequence. To overcome these issues, we developed a modified expression system using a novel phagemid, which we call pScaf. pScaf includes a standard M13 origin of replication (M13 ori) as well as a modified M13 ori which behaves as a terminator of ssDNA synthesis, allowing for packaging and export of the intervening sequence as a custom phage particle, while reducing the fixed-sequence region to only 381 bases.Here, I discuss the steps taken to develop this novel phagemid vector. I also describe the resulting custom scaffolds which we produced using this vector and demonstrate the usefulness of these scaffolds for applications in DNA origami.

The discovery of clustered, regularly interspaced, short palindromic repeats (CRISPR) and the Cas9 RNA-guided nuclease provides unprecedented opportunities to selectively kill specific populations or species of bacteria. However, the use of CRISPR-Cas9 to clear bacterial infections in vivo is hampered by the inefficient delivery of cas9 genetic constructs into bacterial cells. Here, we use a broad-host-range P1-derived phagemid to deliver the CRISPR-Cas9 chromosomal-targeting system into Escherichia coli and the dysentery-causing Shigella flexneri to achieve DNA sequence-specific killing of targeted bacterial cells. We show that genetic modification of the helper P1 phage DNA packaging site (pac) significantly enhances the purity of packaged phagemid and improves the Cas9-mediated killing of S. flexneri cells. We further demonstrate that P1 phage particles can deliver chromosomal-targeting cas9 phagemids into S. flexneri in vivo using a zebrafish larvae infection model, where it significantly reduces the bacterial load and promotes host survival. Our study highlights the potential of combining a P1 bacteriophage-based delivery with the CRISPR chromosomal-targeting system to achieve DNA sequence-specific cell lethality and efficient clearance of bacterial infection.

Antisense oligonucleotides exhibit high potential for use as therapeutic agents. '10-23' DNAzymes are antisense molecules with a high chemical stability and catalytic efficiency. In the present study, we developed a phagemid containing a DNAzyme expression system regulated by two promoters. One of these promoters, pA1, promotes constitutive expression of Moloney murine leukemia virus reverse transcriptase (MoMuLV-RT). The other promoter, plac, regulates transcription of the RNA substrate from which MoMuLV-RT produces the DNAzyme by reverse transcription. The ftsZ DNAzyme was used to validate this expression system in the phagemid, named pDESCP. ftsZ DNAzyme expression altered the morphological pattern of Escherichia coli from a bacillary to filamentous form. In E. coli FtsZ is the primary component of the cell division apparatus, forming a structure known as Z-ring, which is the place of division. It is suggested that the DNAzyme ftsZ is decreasing the translation of this protein. Delivery of pDESCP into F+ strain of E. coli cells, using VCSM13, and the possible insertion of other DNAzymes into the cassette makes this phagemid an important prototype for phage therapy.

A large human natural single‑chain fragment variable (scFv) phage library was constructed based on Cre‑LoxP recombination, and used to successfully identify antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9). The library was derived from 400 blood samples, 30 bone marrow samples, and 10 cord blood samples from healthy donors. Lymphocytes were isolated from each sample and cDNA was synthesized using reverse transcription‑quantitative PCR. Two‑step overlap PCR was then used for scFv synthesis using a LoxP peptide as the linker. The scFv gene was inserted into the phagemid vector pDF by enzymatic digestion and ligation, and then transformed into Escherichia coli (E. coli) SS320 to establish a primary antibody library in the form of scFvs. A primary antibody library consisting of 5x107 peripheral blood and umbilical cord blood sources, as well as a primary antibody library of 5x107 bone marrow samples were obtained. By optimizing the recombination conditions, the primary phage library was used to infect E. coli BS1365 strain (which expresses the Cre enzyme), and a human scFv recombinant library with a size of 1x1011 was obtained through Cre‑LoxP enzyme‑mediated heavy and light chain replacement and recombination. This constructed recombinant library was employed to screen for antibodies against recombinant PCSK9. After four rounds of selection, a fully human antibody (3D2) was identified with a binding affinity of 1.96±1.56ⅹ10‑10 M towards PCSK9. In vitro, the PCSK9/low‑density lipoprotein receptor (LDLR) pathway of Hep‑G2 cells was inhibited by 3D2 treatment, thereby increasing LDL uptake in these cells. In addition, combination treatment with 3D2 and statin was more effective at increasing LDLR levels than treatment with 3D2 or statin alone. Furthermore, 3D2 resulted in a 3‑fold increase in hepatic LDLR levels, and lowered total serum cholesterol by up to 61.5% in vivo. Taken together, these results suggest that the constructed human Cre‑LoxP scFv phage display library can be used to screen fully human scFv, and that 3D2 may serve as a candidate hypolipidemic therapy.

Artificial single-stranded DNA (ssDNA) with user-defined sequences and lengths up to the kilobase range is increasingly needed in mass quantities to realize the potential of emerging technologies such as genome editing and DNA origami. However, currently available biotechnological approaches for mass-producing ssDNA require dedicated, and thus costly, fermentation infrastructure, because of the risk of cross-contaminating manufacturer plants with self-replicating phages. Here we overcome this problem with an efficient, scalable, and cross-contamination-free method for the phage-free biotechnological production of artificial ssDNA with Escherichia coli. Our system utilizes a designed phagemid and an optimized helper plasmid. The phagemid encodes one gene of the M13 phage genome and a freely chosen custom target sequence, while the helper plasmid encodes the other genes of the M13 phage. The phagemid particles produced with this method are not capable of self-replication in the absence of the helper plasmid. This enables cross-contamination-free biotechnological production of ssDNA at any contract manufacturer. Furthermore, we optimized the process parameters to reduce by-products and increased the maximal product concentration up to 83 mg L

Fabs offer an attractive platform for monoclonal antibody discovery/engineering, but library construction can be cumbersome. We report a simple method – Golden Gate assembly with a bi-directional promoter (GBid) – for constructing phage display Fab libraries. In GBid, the constant domains of the Fabs are located in the backbone of the phagemid vector and the library insert comprises only the variable regions of the antibodies and a central bi-directional promoter. This vector design reduces the process of Fab library construction to “scFv-like” simplicity and the double promoter ensures robust expression of both constituent chains. To maximize the library size, the 3 fragments comprising the insert – two variable chains and one bi-directional promoter – are assembled via a 3-fragment overlap extension PCR and the insert is incorporated into the vector via a high-efficiency one-fragment, one-pot Golden Gate assembly. The reaction setup requires minimal preparatory work and enzyme quantities, making GBid highly scalable. Using GBid, we constructed a chimeric chicken-human Fab phage display library comprising 1010 variants targeting the multi-transmembrane protein human CD20 (hCD20). Selection/counter-selection on transfected whole cells yielded hCD20-specific antibodies in four rounds of panning. The simplicity and scalability of GBid makes it a powerful tool for the discovery/engineering of Fabs and IgGs.

Phage display is commonly used to select target-binding antibody fragments from large libraries containing billions of unique antibody clones. In practice, selection outputs are often highly heterogenous, making it desirable to recover sequence information from the selected pool. Next Generation DNA Sequencing (NGS) enables the acquisition of sufficient sequencing reads to cover the pool diversity, however read-lengths are typically too short to capture paired antibody complementarity-determining regions (CDRs), which is needed to reconstruct target-binding antibody fragments. Here, we describe a simple in vitro protocol to bring the DNA encoding the antibody CDRs closer together. The final PCR product referred to as a "CDR strip" is suitable for short read-length NGS. In this method, phagemid ssDNA is recovered from antibody phage display biopanning and used as a template to create a heteroduplex with deletions between CDRs of interest. The shorter strand in the heteroduplex is preferentially PCR amplified to generate a CDR strip that is sequenced using NGS. We have also included a bioinformatics approach to analyze the CDR strip populations so that single antibody clones can be created from paired CDR sequences.

Ubiquitin is a small 8.6 kDa protein that is a core component of the ubiquitin-proteasome system. Consequently, it can bind to a diverse array of proteins with high specificity but low affinity. Through phage display, ubiquitin variants (UbVs) can be engineered such that they exhibit improved affinity over wildtype ubiquitin and maintain binding specificity to target proteins. Phage display utilizes a phagemid library, whereby the pIII coat protein of a filamentous M13 bacteriophage (chosen because it is displayed externally on the phage surface) is fused with UbVs. Specific residues of human wildtype ubiquitin are soft and randomized (i.e., there is a bias towards to native wildtype sequence) to generate UbVs so that deleterious changes in protein conformation are avoided while introducing the diversity necessary for promoting novel interactions with the target protein. During the phage display process, these UbVs are expressed and displayed on phage coat proteins and panned against a protein of interest. UbVs that exhibit favorable binding interactions with the target protein are retained, whereas poor binders are washed away and removed from the library pool. The retained UbVs, which are attached to the phage particle containing the UbV's corresponding phagemid, are eluted, amplified, and concentrated so that they can be panned against the same target protein in another round of phage display. Typically, up to five rounds of phage display are performed, during which a strong selection pressure is imposed against UbVs that bind weakly and/or promiscuously so that those with higher affinities are concentrated and enriched. Ultimately, UbVs that demonstrate higher specificity and/or affinity for the target protein than their wildtype counterparts are isolated and can be characterized through further experiments.

Simple Summary Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants. Current efforts are directed towards the global control and eradication of PPRV, an initiative of the World Organisation for Animal Health and Food and the Agriculture Organisation of the United Nations. A plethora of diagnostic tools for PPR were primarily developed for livestock. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems, and in complex field situations. Recent studies confirmed that single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have proven to be a powerful tool in diagnostics and therapeutics due to their unique properties, such as small size and strong antigen-binding affinity. Therefore, the main objective of this study was to generate PPRV-reactive nanobodies in order to set a pace for the development of diagnostic and possibly therapeutic nanobodies in the future. Initially, a strategy was developed whereby an alpaca was immunized with PPRV in order to raise an affinity-matured immune response, from which an immune nanobody library was constructed. Following phage display, nine nanobodies that specifically recognise PPRV were identified on enzyme-linked immunosorbent assay. This study has generated PPRV-reactive nanobodies and have significant implications in the development of cost-effective diagnostic tools in context with the planned eradication of PPR in the world. Abstract Peste des petits ruminants virus (PPRV) causes a highly devastating disease, peste des petits ruminants (PPR) of sheep and goats, that threatens food security, small ruminant production, and the conservation of wild small ruminants in many developing countries, especially in Africa. Robust serological and molecular diagnostic tools are available to detect PPRV infection, but they were mainly developed for domestic sheep and goats. The presence of a wide host range for PPRV does present serological diagnostic challenges. New innovative diagnostic tools are needed to detect PPRV in atypical hosts (e.g., Camelidae, Suidae, and Bovinae), in wildlife ecosystems and in complex field situations. Interestingly, single-domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies have emerged as a new hope in the development of accurate, rapid, and cost-effective diagnostic tools in veterinary and biomedical fields that are suitable for low-income countries. The main objective of this study was to construct an immune nanobody library to retrieve PPRV-reactive nanobodies that enable the development of diagnostic and therapeutic nanobodies in the future. Here, a strategy was developed whereby an alpaca (Vicugna pacos) was immunized with a live attenuated vaccine strain (PPRV/N/75/1) to raise an affinity-matured immune response in the heavy-chain-only antibody classes. The nanobody gene repertoire was engineered in pMECS-GG phagemid, whereby a ccdB gene (encoding a lethal protein) was substituted by the nanobody gene. An immune nanobody library with approximately sixty-four million independent transformants was constructed, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and biopanning, nine nanobodies that specifically recognise completely inactivated PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in rapidly identifying PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPR infection.

[Background] The hemolysin (Hly) secretion system of E. coli allows the one-step translocation of hemolysin A (HlyA) from the bacterial cytoplasm to the extracellular medium, without a periplasmic intermediate. In this work, we investigate whether the Hly secretion system of E. coli is competent to secrete a repertoire of functional single-domain VHH antibodies (nanobodies, Nbs), facilitating direct screening of VHH libraries and the purification of selected Nb from the extracellular medium., [Results] We employed a phagemid library of VHHs obtained by immunization of a dromedary with three protein antigens from enterohemorrhagic E. coli (EHEC), namely, the extracellular secreted protein A (EspA), the extracellular C-terminal region of Intimin (Int280), and the translocated intimin receptor middle domain (TirM). VHH clones binding each antigen were enriched and amplified by biopanning, and subsequently fused to the C-terminal secretion signal of HlyA to be expressed and secreted in a E. coli strain carrying the Hly export machinery (HlyB, HlyD and TolC). Individual E. coli clones were grown and induced in 96-well microtiter plates, and the supernatants of the producing cultures directly used in ELISA for detection of Nbs binding EspA, Int280 and TirM. A set of Nb sequences specifically binding each of these antigens were identified, indicating that the Hly system is able to secrete a diversity of functional Nbs. We performed thiol alkylation assays demonstrating that Nbs are correctly oxidized upon secretion, forming disulphide bonds between cysteine pairs despite the absence of a periplasmic intermediate. In addition, we show that the secreted Nb-HlyA fusions can be directly purified from the supernatant of E. coli cultures, avoiding cell lysis and in a single affinity chromatography step., [Conclusions] Our data demonstrate the Hly secretion system of E. coli can be used as an expression platform for screening and purification of Nb binders from VHH repertories., We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Work in the lab of LAF is partially supported by the Grants BIO2017-89081-R (Agencia Española de Investigación AEI/MICIU/FEDER, EU), ERC-2012-ADG_20120314 (European Research Council, EU) and LCCM_P59474 (Wellcome Trust, UK).

Phage display links phenotype of displayed polypeptides with DNA sequence in phage genome and offers a universal method for discovery of proteins with novel properties. Injection of phage-displayed libraries in living organisms further provides a unique and powerful approach to optimize biochemical, pharmacological and biological properties of the displayed peptides, antibodies and other proteins in vivo. However, over 60% of the proteome is comprised of multi-domain proteins, and display of large multi-subunit proteins on phages remains a challenge. Majority of protein display systems are based on monovalent phagemid constructs but methods for robust display of multiple copies of large proteins are scarce. Here, we describe a DNA-encoded display of a ∼200 kDa tetrameric protein tetrameric L-asparaginase on M13 phage produced by ligation of SpyCatcher-Asparaginase fusion (ScA) to prospectively barcoded phage clones displaying SpyTag peptide. Starting from the SpyTag display on p3 minor coat protein or p8 major coat protein yielded constructs with five copies of ScA displayed on p3 (ScA5-phage) and 50 copies of ScA on p8 protein (ScA50-phage). ScA remained active after conjugation. It could be easily produced directly from lysates of bacteria that express ScA. Display constructs of different valency can be injected into mice and analyzed by deep-sequencing of the DNA barcodes associated phage clones. In these multiplexed studies, we observed a density-dependent clearance rate in vivo. A known clearance mechanism of L-asparaginase is endocytosis by phagocytic cells. Our observations, thus, link the increase in density of the displayed protein with the increased rate of the endocytosis by cells in vivo. In conclusion, we demonstrate that a multivalent display of L-asparaginase on phage could be used to study the circulation life of this protein in vivo and such approach opens the possibility to use DNA sequencing to investigate multiplexed libraries of other multi-subunit proteins in vivo.Abstract Graphic

This study reports on the characterization of a Klebsiella pneumoniae clinical isolate showing high-level resistance to ceftazidime-avibactam associated with the production of KPC-53, a KPC-3 variant exhibiting a Leu167Glu168 duplication in the Ω-loop and a loss of carbapenemase activity. Whole-genome sequencing (WGS) revealed the presence of two copies of bla KPC-53 , located on a pKpQIL-like plasmid and on a plasmid prophage of the Siphoviridae family, respectively.

Here, we provide a protocol to generate synthetic nanobodies, known as sybodies, against any purified protein or protein complex within a 3-week period. Unlike methods that require animals for antibody generation, sybody selections are carried out entirely in vitro under controlled experimental conditions. This is particularly relevant for the generation of conformation-specific binders against labile membrane proteins or protein complexes and allows selections in the presence of non-covalent ligands. Sybodies are especially suited for cases where binder generation via immune libraries fails due to high sequence conservation, toxicity or insufficient stability of the target protein. The procedure entails a single round of ribosome display using the sybody libraries encoded by mRNA, followed by two rounds of phage display and binder identification by ELISA. The protocol is optimized to avoid undesired reduction in binder diversity and enrichment of non-specific binders to ensure the best possible selection outcome. Using the efficient fragment exchange (FX) cloning method, the sybody sequences are transferred from the phagemid to different expression vectors without the need to amplify them by PCR, which avoids unintentional shuffling of complementary determining regions. Using quantitative PCR (qPCR), the efficiency of each selection round is monitored to provide immediate feedback and guide troubleshooting. Our protocol can be carried out by any trained biochemist or molecular biologist using commercially available reagents and typically gives rise to 10-30 unique sybodies exhibiting binding affinities in the range of 500 pM-500 nM.

Objective To determine the effect of exposure to remnants of a phagemid-containing E. coli, killed by treatment with a propanol-based hand rub, on antimicrobial resistance in E. coli isolates. Methods An in vitro model was developed in which a clinical E. coli isolate (EUR1) was exposed to remnants of an E. coli K-12 strain containing a phagemid (pBS-E12) strain treated with Sterillium®. A series of 200 experiments was performed using this in vitro model. As a control, a series of 400 experiments was performed where the EUR1 was exposed either to the remnants of an E. coli K-12 strain (not containing a phagemid) (E12) treated with Sterillium® (n = 200) or to dried Sterillium® only (n = 200). The number of experiments that showed growth of an amoxicillin-resistant EUR1 isolate was evaluated in all three groups. An additional 48 experiments were performed in which a different clinical E. coli isolate (EUR2) was exposed to remnants of the pBS-E12 treated with Sterillium®. Whole-genome sequencing and phenotypic testing for AmpC beta-lactamase production was performed to investigate the mechanism behind this resistance development. Results In 22 (11.0%) of 200 experiments in which the EUR1 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained, as opposed to only 2 (1.0%) of 200 experiments involving the exposure of the EUR1 to Sterillium® only (risk difference: 10.0%; 95% CI 5.4–14.6%)) and 1 (0.5%) of 200 experiments involving the exposure of the EUR1 isolate to the remnants of the phagemid-free E12 (risk difference: 10.5%; 95% CI 6.1–14.9%). In 1 (2.1%) of the 48 experiments in which the EUR2 isolate was exposed to remnants of a pBS-E12 an amoxicillin-resistant mutant isolate was obtained. The development of resistance in all experiments was due to mutations in the promoter/attenuator region of the chromosomal AmpC beta-lactamase (cAmpC) gene leading to cAmpC hyperproduction. Conclusion Exposure of an E. coli isolate to another phagemid-containing E. coli that was treated with propanol-based hand rub increased the development of amoxicillin resistance. Although phagemids are cloning vectors that are not present in clinical isolates, this finding may have implications for hand disinfection practices in healthcare facilities.

As a popular assembly technique, DNA origami offered a simple way to fold long, single-stranded (ss) DNA molecules into arbitrary two-dimensional (2D) shapes. To design a desired shape, a 7-kilobase M13mp18 genomic DNA was raster filled into the shape as a scaffold, and over hundreds short oligonucleotide staple strands were carefully chosen and added in excessive molar ratios to hold the scaffold in place. The assembly of a desired 2D shape with a diameter of ~100 nm can be achieved in a simple annealing step in hours. Due to its convenience during preparation and the large addressable surface area of the assembled shapes, DNA origami has been rapidly explored as templates for many types of biomaterial and biomedical applications. However, a few limitations hinder the further development of this assembly technique. One is about the relatively high cost: When it comes to large scale preparation (e.g., gram-level) for potential drug evaluation in animals, the requirement of large scale of hundreds of synthetic DNA strands usually brings about bills near to 0.1−1 Million Chinese Yuan. Another issue is the leftover of the excess of the staple strands: They are hardly to be completely removed from the desired DNA structures, which may cause unwanted side-effects during biomedical applications. If a desired shape could be assembled with only one ssDNA in a high yield, the second issue would not exist anymore. As early as in 2007, the Yans group reported that the DNA tile with odd number of the paranemic crossover could be assembled by one ssDNA. Later on, several groups found that the tetrahedron and prism-like scaffold objects could also be assembled with an ssDNA. However these DNA nanostructures created by a ssDNA (~hundred bases in length) were relatively small, with diameters 10 nm. The challenge to create large shapes with only one ssDNA is that in the one polymer folding pathway, the crossovers that bundle many helical domains to form the shapes would inevitably encounter the topological knotting issue, which set the shape formation into many kinetic traps. About ten years later, recently the Yan and Yins group developed the ssDNA origami technique as a derivative of the traditional DNA origami by integrating all sequences required by a desired shape into one long single strand. The key innovation is to use partially complemented double-stranded DNA and parallel crossover cohesion to construct a knot-free structure that can be folded smoothly from a single strand. The removal of the excess of staple strands yet still possessing a high yield ( > 90%) of the desired shapes and the adaptiveness to single-stranded RNA origami hint a promising future of this new technique in biomedical and biomaterial applications. Moreover, the recent development of single-stranded DNA preparation, for example, extraction of single-stranded genomic DNA with customized sequences up to 30000 bases from the helper phage/phagemid system, enabled the acquisition of gram-level of long single-stranded DNA in a cost-effective manner (~10 Thousand Chinese Yuan) and offered a solid foundation for large scale assembly of single-stranded DNA origami. The settlement of the two major issues in the traditional DNA origami should pave the way for applying DNA origami-based nanostructures in clinical tests. In this paper, we summarized the research progress of DNA self-assembly with one long single strand and the current ways to prepare long single-stranded DNAs, and finished with our thoughts on the future of the new single-stranded DNA origami technique.

Phage display of combinatorial antibody libraries is a versatile tool in the field of antibody engineering, with diverse applications including monoclonal antibody (mAb) discovery, affinity maturation, and humanization. To improve the selection efficiency of antibody libraries, we developed a new phagemid display system that addresses the complication of bald phage propagation. The phagemid facilitates the biotinylation of fragment of antigen binding (Fab) antibody fragments displayed on phage via Sortase A catalysis and the subsequent enrichment of Fab-displaying phage during selections. In multiple contexts, this selection approach improved the enrichment of target-reactive mAbs by depleting background phage. Panels of cancer cell line-reactive mAbs with high diversity and specificity were isolated from a naïve chimeric rabbit/human Fab library using this approach, highlighting its potential to accelerate antibody engineering efforts and to empower concerted antibody drug and target discovery.

Early efforts to genetically engineer biological systems were restricted to relatively crude cut and paste operations, with the sequence of even model organisms' genomes inaccessible for detailed study. Exponential improvement in our ability to both read and write DNA sequences has dramatically altered the landscape of genetic engineering, rendering de novo synthesis of entire genomes possible. However, our ability to design functional systems has not kept pace with our ability to fabricate them. The field of synthetic biology seeks to address this shortcoming and provide a theoretical framework for rationally manipulating biological systems. Of particular interest is exploration of how core engineering principles such as modularity, abstraction, and standardization can be applied to the development of genetic systems. Despite considerable recent progress toward answering that question, imperfect and incomplete knowledge about biological systems necessitates exploration of numerous variants to craft even relatively simple systems. In this work, we first explore development of biological systems that make engineering biology easier, then pursue development of a complex system for a demanding application to probe the limits of our design capability, and conclude with a discussion of key challenges in designing biological systems and how to address them.Motivated by the observation that tuning the expression of system components is often essential to achieving desired functions, we begin by describing development of a system for rapidly prototyping genetic constructs to determine an optimal expression level. Although several methods exist for manipulating the transcription or translation of a desired gene, they require fabrication of numerous distinct variants. To work around this limitation, we instead explore manipulation of plasmid copy number and develop a set of strains that are capable of maintaining the same plasmid at a desired level in the range of 1-250 copies per cell. We demonstrate that this system can be applied to rapidly find the optimal expression level for a model biosynthetic pathway, regardless of the transcriptional activity of the pathway. Recognizing that fabricating and assaying distinct variants is in some cases essential, we then examined the problem of enabling high-throughput manipulation of DNA. Existing methods are limited by difficult to automate operations, such as centrifugation, application of a vacuum, or gel electrophoresis. Development of alternative methods that only require liquid handling operations would enable utilization of very high throughput automation platforms. We therefore engineer a P1 phage-based system for transferring plasmids between cells using only liquid handling operations. After establishing exogenous control of phage lysis through addition of a small molecule inducer, we incorporate phage cis elements that enable desired DNA to be transferred 1600-fold more efficiently than other cellular DNA. The system is capable of transferring large libraries of DNA up to 25 kilobases in length at small volumes. This provides an important first step toward development of a highly automatable suite of tools for biological engineering.Biological engineers ultimately want to manipulate biological systems to solve human specified problems. Although a number of potential applications of synthetic biology have been described, most projects rely on relatively simple designs to achieve a desired outcome. To identify bottlenecks in the development of more complex systems, we investigate engineering a laboratory strain of E. coli to localize to, invade, and kill cancer cells. After attempting to transfer capsular polysaccharide synthesis clusters to a laboratory strain to improve bacterial survival in an animal model, we generate a strain capable of delivering a cytotoxic ribonuclease to and subsequently killing cultured cancer cells. Although the strain did not inhibit tumor growth in an animal model, the lessons learned during the execution of this and other projects guided our thinking about current bottlenecks and the path forward. We conclude with a discussion of the potential for modular design to enable routine development of complex biological systems, and identify six failure modes that hinder current efforts. By architecting software to codify existing biological knowledge and investigating the basis of important phenomena such as load and stress, we can harness the power of improved DNA sequencing and synthesis capabilities to build novel, useful biological systems.