Mutation of the peptide-regulated transcription factor ComR for amidated peptide specificity and heterologous function in Lactiplantibacillus plantarum WCFS1.

There is a growing interest in the use of probiotic bacteria as biosensors for the detection of disease. However, there is a lack of bacterial receptors developed for specific disease biomarkers. Here, we have investigated the use of the peptide-regulated transcription factor ComR from Streptococcus spp. for specific peptide biomarker detection. ComR exhibits a number of attractive features that are potentially exploitable to create a biomolecular switch for engineered biosensor circuitry within the probiotic organism Lactiplantibacillus plantarum WCFS1. Through iterative design-build-test cycles, we developed a genomically integrated, ComR-based biosensor circuit that allowed WCFS1 to detect low nanomolar concentrations of ComR's cognate peptide XIP. By screening a library of ComR proteins with mutant residues substituted at the K100 position, we identified mutations that increased the specificity of ComR toward an amidated version of its cognate peptide, demonstrating the potential for ComR to detect this important class of biomarker.IMPORTANCEUsing bacteria to detect disease is an exciting possibility under active study. Detecting extracellular peptides with specific amino acid sequences would be particularly useful as these are important markers of health and disease (biomarkers). In this work, we show that a probiotic bacteria (Lactiplantibacillus plantarum) can be genetically engineered to detect specific extracellular peptides using the protein ComR from Streptococcus bacteria. In its natural form, ComR allowed the probiotic bacteria to detect a specific peptide, XIP. We then modified XIP to be more like the peptide biomarkers found in humans and engineered ComR so that it activated with this modified XIP and not the original XIP. This newly engineered ComR also worked in the probiotic bacteria, as expected. This suggests that with additional engineering, ComR might be able to activate with human peptide biomarkers and be used by genetically engineered probiotic bacteria to better detect disease.

On-chip dielectrophoretic recovery and detection of a lactate sensing probiotic from model human saliva.

Early detection has led to increased survival for multiple cancers; however, the 5-year survival rate of oral carcinoma (OC) has remained at 40% for the last several decades. Screening for OC is routinely done via visual examinations, followed by tissue biopsy and laboratory testing. Point-of-care testing would be a more convenient and widely available alternative for at-risk individuals. Increased lactate production is a hallmark of many head-and-neck tumors, due to the Warburg Effect, where tumor cells favor glycolysis in the place of oxidative phosphorylation. To detect excess lactate, we have modified the commensal bacterium Escherichia coli Nissle 1917 to express fluorescent reporter genes in response to extracellular lactate. Administering this commensal as a mouth wash and subsequently collecting saliva for the detection of the reporter may allow for noninvasive, early detection of cancerous lesions in at-risk individuals. Furthermore, we demonstrate a new on-chip electrokinetic technique to recover these probiotic probes from model saliva fluid to improve the detection of reporter gene activation.

Turning antibodies off and on again using a covalently tethered blocking peptide.

In their natural form, antibodies are always in an "on-state" and are capable of binding to their targets. This leads to undesirable interactions in a wide range of therapeutic, analytical, and synthetic applications. Modulating binding kinetics of antibodies to turn them from an "off-state" to an "on-state" with temporal and spatial control can address this. Here we demonstrate a method to modulate binding activity of antibodies in a predictable and reproducible way. We designed a blocking construct that uses both covalent and non-covalent interactions with the antibody. The construct consisted of a Protein L protein attached to a flexible linker ending in a blocking-peptide designed to interact with the antibody binding site. A mutant Protein L was developed to enable photo-triggered covalent crosslinking to the antibody at a specific location. The covalent bond anchored the linker and blocking peptide to the antibody light chain keeping the blocking peptide close to the antibody binding site. This effectively put the antibody into an "off-state". We demonstrate that protease-cleavable and photocleavable moieties in the tether enable controlled antibody activation to the "on-state" for anti-FLAG and cetuximab antibodies. Protein L can bind a range of antibodies used therapeutically and in research for wide applicability.

Enzymes Photo-Cross-Linked to Live Cell Receptors Retain Activity and EGFR Inhibition after Both Internalization and Recycling.

In this work, we show that a prodrug enzyme covalently photoconjugated to live cell receptors survives endosomal proteolysis and retains its catalytic activity over multiple days. Here, a fusion protein was designed with both an antiepidermal growth factor receptor (EGFR) affibody and the prodrug enzyme cytosine deaminase, which can convert prodrug 5-fluorocytosine to the anticancer drug 5-fluorouracil. A benzophenone group was added at a site-specific mutation within the affibody, and the fusion protein was selectively photoconjugated to EGFR receptors expressed on membranes of MDA-MB-468 breast cancer cells. The fusion protein was next labeled with two dyes for tracking uptake: AlexaFluor 488 and pH-sensitive pHAb. Flow cytometry showed that fusion proteins photo-cross-linked to EGFR first underwent receptor-mediated endocytosis within 12 h, followed by recycling back to the cell membrane within 24 h. These findings were also confirmed by confocal microscopy. The unique cross-linking of the affibody-enzyme fusion proteins was utilized for two anticancer treatments. First, the covalent linking of the protein to the EGFR led to inhibition of ERK signaling over a two-day period, whereas conventional antibody therapy only led to 6 h of inhibition. Second, when the affibody-CodA fusion proteins were photo-cross-linked to EGFR overexpressed on MDA-MB-468 breast cancer cells, prodrug conversion was found even 48 h postincubation without any apparent decrease in cell killing, while without photo-cross-linking no cell killing was observed 8 h postincubation. These studies show that affinity-mediated covalent conjugation of the affibody-enzymes to cell receptors allows for prolonged expression on membranes and retained enzymatic activity without genetic engineering.

Anti-EGFR Affibodies with Site-Specific Photo-Cross-Linker Incorporation Show Both Directed Target-Specific Photoconjugation and Increased Retention in Tumors.

A significant challenge for solid tumor treatment is ensuring that a sufficient concentration of therapeutic agent is delivered to the tumor site at doses that can be tolerated by the patient. Biomolecular targeting can bias accumulation in tumors by taking advantage of specific interactions with receptors overexpressed on cancerous cells. However, while antibody-based immunoconjugates show high binding to specific cells, their low dissociation constants ( KD) and large Stokes radii hinder their ability to penetrate deep into tumor tissue, leading to incomplete cell killing and tumor recurrence. To address this, we demonstrate the design and production of a photo-cross-linkable affibody that can form a covalent bond to epidermal growth factor receptor (EGFR) under near UV irradiation. Twelve cysteine mutations were created of an EGFR affibody and conjugated with maleimide-benzophenone. Of these only one exhibited photoconjugation to EGFR, as demonstrated by SDS-PAGE and Western blot. Next this modified affibody was shown to not only bind EGFR expressing cells but also show enhanced retention in a 3D tumor spheroid model, with minimal loss up to 24 h as compared to either unmodified EGFR-binding affibodies or nonbinding, photo-cross-linkable affibodies. Finally, in order to show utility of photo-cross-linking at clinically relevant wavelengths, upconverting nanoparticles (UCNPs) were synthesized that could convert 980 nm light to UV and blue light. In the presence of UCNPs, both direct photoconjugation to EGFR and enhanced retention in tumor spheroids could be obtained using near-infrared illumination. Thus, the photoactive affibodies developed here may be utilized as a platform technology for engineering new therapy conjugates that can penetrate deep into tumor tissue and be retained long enough for effective tumor therapy.

TiO2-Capped Gold Nanorods for Plasmon-Enhanced Production of Reactive Oxygen Species and Photothermal Delivery of Chemotherapeutic Agents.

Near infrared (NIR)-absorbing noble metal nanostructures are being extensively studied as theranostic agents, in particular for photoacoustic imaging and photothermal therapy. Because of the electric field enhancement at the tips of anisotropic metal nanostructures, positioning photoactive species at these sites can lead to increased energy absorption. Herein, we show the site-specific placement of NIR-active photosensitizers at the ends of gold nanorods (AuNRs) by growing porous TiO2 caps. The surface plasmon resonance of the AuNRs was carefully tuned to overlap with the exciton absorption of indocyanine green (ICG), a NIR photosensitizer with low quantum yields and poor photostability. In conjugating high amounts of ICG to the TiO2 caps, increased amounts of singlet oxygen (1O2) were generated as compared to when ICG was attached to sidewalls of the AuNRs. Because the AuNRs also cause local increases in temperature upon NIR excitation, DNA strands were next attached to the AuNRs sidewalls and loaded with doxorubicin (DOX). We found that the synergistic effect of increased 1O2 and photothermal-induced drug delivery led to significant improvements in tumor cell killing. This work demonstrates that with careful design over hybrid nanostructure synthesis, higher levels of tumor therapy may be achieved.

DNA-Assembled Core-Satellite Upconverting-Metal-Organic Framework Nanoparticle Superstructures for Efficient Photodynamic Therapy.

DNA-mediated assembly of core-satellite structures composed of Zr(IV)-based porphyrinic metal-organic framework (MOF) and NaYF4 ,Yb,Er upconverting nanoparticles (UCNPs) for photodynamic therapy (PDT) is reported. MOF NPs generate singlet oxygen (1 O2 ) upon photoirradiation with visible light without the need for additional small molecule, diffusional photosensitizers such as porphyrins. Using DNA as a templating agent, well-defined MOF-UCNP clusters are produced where UCNPs are spatially organized around a centrally located MOF NP. Under NIR irradiation, visible light emitted from the UCNPs is absorbed by the core MOF NP to produce 1 O2 at significantly greater amounts than what can be produced from simply mixing UCNPs and MOF NPs. The MOF-UCNP core-satellite superstructures also induce strong cell cytotoxicity against cancer cells, which are further enhanced by attaching epidermal growth factor receptor targeting affibodies to the PDT clusters, highlighting their promise as theranostic photodynamic agents.

Isothermal rolling circle amplification of virus genomes for rapid antigen detection and typing.

In this work, isothermal rolling circle amplification (RCA) of the multi-kilobase genome of engineered filamentous bacteriophage is used to report the presence and identification of specific protein analytes in solution. First, bacteriophages were chosen as sensing platforms because peptides or antibodies that bind medically relevant targets can be isolated through phage display or expressed as fusions to their p3 and p8 coat proteins. Second, the circular, single-stranded genome contained within the phage serves as a natural large DNA template for a RCA reaction to rapidly generate exponential amounts of double stranded DNA in a single isothermal step that can be easily detected using low-cost fluorescent nucleic acid stains. Amplifying the entire phage genome also provides high detection sensitivities. Furthermore, since the sequence of the viral DNA can be easily modified with multiple restriction enzyme sites, a simple DNA digest can be applied to detect and identify multiple antigens simultaneously. The methods developed here will lead to protein sensors that are highly scalable to produce, can be run without complex biological equipment and do not require the use of multiple antibodies or high-cost fluorescent DNA probes or nucleotides.

Creating highly amplified enzyme-linked immunosorbent assay signals from genetically engineered bacteriophage.

For early detection of many diseases, it is critical to be able to diagnose small amounts of biomarkers in blood or serum. One of the most widely used sensing assays is the enzyme-linked immunosorbent assay (ELISA), which typically uses detection monoclonal antibodies conjugated to enzymes to produce colorimetric signals. To increase the overall sensitivities of these sensors, we demonstrate the use of a dually modified version of filamentous bacteriophage Fd that produces significantly higher colorimetric signals in ELISAs than what can be achieved using antibodies alone. Because only a few proteins at the tip of the micron-long bacteriophage are involved in antigen binding, the approximately 4000 other coat proteins can be augmented-by either chemical functionalization or genetic engineering-with hundreds to thousands of functional groups. In this article, we demonstrate the use of bacteriophage that bear a large genomic fusion that allows them to bind specific antibodies on coat protein 3 (p3) and multiple biotin groups on coat protein 8 (p8) to bind to avidin-conjugated enzymes. In direct ELISAs, the anti-rTNFα (recombinant human tumor necrosis factor alpha)-conjugated bacteriophage show approximately 3- to 4-fold gains in signal over that of anti-rTNFα, demonstrating their use as a platform for highly sensitive protein detection.

Engineered calcium-precipitable restriction enzyme.

We have developed a simple system for tagging and purifying proteins. Recent experiments have demonstrated that RTX (Repeat in Toxin) motifs from the adenylate cyclase toxin gene (CyaA) of B. pertussis undergo a conformational change upon binding calcium, resulting in precipitation of fused proteins and making this method a viable alternative for bioseparation. We have designed an iGEM Biobrick comprised of an RTX tag that can be easily fused to any protein of interest. In this paper, we detail the process of creating an RTX tagged version of the restriction enzyme EcoRI and describe a method for expression and purification of the functional enzyme.

Serial and parallel Si, Ge, and SiGe direct-write with scanning probes and conducting stamps.

Precise materials integration in nanostructures is fundamental for future electronic and photonic devices. We demonstrate Si, Ge, and SiGe nanostructure direct-write with deterministic size, geometry, and placement control. The biased probe of an atomic force microscope (AFM) reacts diphenylsilane or diphenylgermane to direct-write carbon-free Si, Ge, and SiGe nano and heterostructures. Parallel direct-write is available on large areas by substituting the AFM probe with conducting microstructured stamps. This facile strategy can be easily expanded to a broad variety of semiconductor materials through precursor selection.