Svetlana Harbaugh

Hydrogen-bonded LbL shells for living cell surface engineering

We report on the design of cytocompatible synthetic shells from highly permeable, hydrogen-bonded multilayers for cell surface engineering with preservation of long-term cell functioning. In contrast to traditional polyelectrolyte layer-by-layer (LbL) systems, shells suggested here are based on hydrogen bonding allowing gentle cell encapsulation using non-toxic, non-ionic and biocompatible components such as poly(N-vinylpyrrolidone) (PVPON) and tannic acid (TA) which were earlier exploited on abiotic surfaces but never assembled on cell surfaces. Here, we demonstrate that these LbL shells with higher diffusion facilitate outstanding cell survivability reaching 79% in contrast to only 20% viability level achieved with ionically paired coatings. We suggest that the drastic increase in cell viability and preservation of cell functioning after coating with synthetic shell stems from the minimal exposure of the cells to toxic polycations and high shell permeability.

pH-Responsive Layer-by-Layer Nanoshells for Direct Regulation of Cell Activity

Saccharomyces cerevisiae yeast cells encapsulated with pH-responsive synthetic nanoshells from lightly cross-linked polymethacrylic acid showed a high viability rate of around 90%, an indication of high biocompatibility of synthetic pH-responsive shells. We demonstrated that increasing pH above the isoelectric point of the polymer shell leads to a delay in growth rate; however, it does not affect the expression of enhanced green fluorescent protein. We suggest that progressive ionization and charge accumulation within the synthetic shells evoke a structural change in the outer shells which affect the membrane transport. This change facilitates the ability to manipulate growth kinetics and functionality of the cells with the surrounding environment. We observed that hollow layer-by layer nanoshells showed a remarkable degree of reversible swelling/deswelling over a narrow pH range (pH 5.0-6.0), but their assembly directly on the cell surface resulted in the suppression of large dimensional changes. We suggest that the variation in surface charges caused by deprotonation/protonation of carboxylic groups in the nanoshells controlled cell growth and cell function, which can be utilized for external chemical control of cell-based biosensors.

Truly nonionic polymer shells for the encapsulation of living cells.

Engineering surfaces of living cells with natural or synthetic compounds can mediate intercellular communication and provide a protective barrier from hostile agents. We report on truly nonionic hydrogen-bonded LbL coatings for cell surface engineering. These ultrathin, highly permeable polymer membranes are constructed on living cells without the cationic component typically employed to increase the stability of LbL coatings. Without the cytotoxic cationic PEI pre-layer, the viability of encapsulated cells drastically increases to 94%, in contrast to 20% viability in electrostatically-bonded LbL shells. Moreover, the long-term growth of encapsulated cells is not affected, thus facilitating efficient function of protected cells in hostile environment.

Silk Macromolecules with Amino Acid–Poly(Ethylene Glycol) Grafts for Controlling Layer-by-Layer Encapsulation and Aggregation of Recombinant Bacterial Cells

This study introduces double-brush designs of functionalized silk polyelectrolytes based upon regenerated silk fibroin (SF), which is modified with poly-L-lysine (SF-PLL), poly-L-glutamic acid (SF-PGA), and poly(ethylene glycol) (PEG) side chains with different grafting architecture and variable amino acid-PEG graft composition for cell encapsulation. The molecular weight of poly amino acids (length of side chains), molecular weight and degree of PEG grafting (D) were varied in order to assess the formation of cytocompatible and robust layer-by-layer (LbL) shells on two types of bacterial cells (Gram-negative and Gram-positive bacteria). We observed that shells assembled with charged polycationic amino acids adversely effected the properties of microbial cells while promoting the formation of large cell aggregates. In contrast, hydrogen-bonded shells with high PEG grafting density were the most cytocompatible, while promoting formation of stable colloidal suspensions of individual cell encapsulates. The stability to degradation of silk shells (under standard cell incubation procedure) was related to the intrinsic properties of thermodynamic bonding forces, with shells based on electrostatic interactions having stronger resistance to deterioration compared to pure hydrogen-bonded silk shells. By optimizing the charge density of silk polyelectrolytes brushes, as well as the length and the degree of PEG side grafts, robust and cytocompatible cell coatings were engineered that can control aggregation of cells for biosensor devices and other potential biomedical applications.

Development of a 2,4-Dinitrotoluene-Responsive Synthetic Riboswitch in E. coli Cells

Riboswitches are RNA sequences that regulate expression of associated downstream genes in response to the presence or absence of specific small molecules. A novel riboswitch that activates protein translation in E. coli cells in response to 2,4-dinitrotoluene (DNT) has been engineered. A plasmid library was constructed by incorporation of 30 degenerate bases between a previously described trinitrotoluene aptamer and the ribosome binding site. Screening was performed by placing the riboswitch library upstream of the Tobacco Etch Virus (TEV) protease coding sequence in one plasmid; a second plasmid encoded a FRET-based construct linked with a peptide containing the TEV protease cleavage site. Addition of DNT to bacterial culture activated the riboswitch, initiating translation of TEV protease. In turn, the protease cleaved the linker in the FRET-based fusion protein, causing a change in fluorescence. This new riboswitch exhibited a 10-fold increase in fluorescence in the presence of 0.5 mM DNT compared to the system without target.

Cell Surface Engineering with Edible Protein Nanoshells

Natural protein (silk fibroin) nanoshells are assembled on the surface of Saccharomyces cerevisiae yeast cells without compromising their viability. The nanoshells facilitate initial protection of the cells and allow them to function in encapsulated state for some time period, afterwards being completely biodegraded and consumed by the cells. In contrast to a traditional methanol treatment, the gentle ionic treatment suggested here stabilizes the shell silk fibroin structure but does not compromise the viability of the cells, as indicated by the fast response of the encapsulated cells, with an immediate activation by the inducer molecules. Extremely high viability rates (up to 97%) and preserved activity of encapsulated cells are facilitated by cytocompatibility of the natural proteins and the formation of highly porous shells in contrast to traditional polyelectrolyte-based materials. Moreover, in a high contrast to traditional synthetic shells, the silk proteins are biodegradable and can be consumed by cells at a later stage of growth, thus releasing the cells from their temporary protective capsules. These on-demand encapsulated cells can be considered a valuable platform for biocompatible and biodegradable cell encapsulation, controlled cell protection in a synthetic environment, transfer to a device environment, and cell implantation followed by biodegradation and consumption of protective protein shells.

Orthogonal Cell-Based Biosensing: Fluorescent, Electrochemical, and Colorimetric Detection with Silica-Immobilized Cellular Communities Integrated with an ITO–Glass/Plastic Laminate Cartridge

This is the first report of a living cell-based environmental sensing device capable of generating orthogonal fluorescent, electrochemical, and colorimetric signals in response to a single target analyte in complex media. Orthogonality is enabled by use of cellular communities that are engineered to provide distinct signals in response to the model analyte. Coupling these three signal transduction methods provides additional and/or complementary data regarding the sample which may reduce the impact of interferants and increase confidence in the sensors output. Long-term stability of the cells was addressed via 3D entrapment within a nanostructured matrix derived from glycerated silicate, which allows the device to be sealed and stored under dry, ambient conditions for months with significant retention in cellular activity and viability (40% viability after 60 days). Furthermore, the first co-entrapment of eukaryotic and bacterial cells in a silica matrix is reported, demonstrating multianalyte biodetection by mixing disparate cell lines at intimate proximities which remain viable and responsive. These advances in cell-based biosensing open intriguing opportunities for integrating living cells with nanomaterials and macroscale systems.

Bacterial Sunscreen: Layer-by-Layer Deposition of UV-Absorbing Polymers on Whole-Cell Biosensors

UV-protective coatings on live bacterial cells were created from the assembly of cationic and UV-absorbing anionic polyelectrolytes using layer-by-layer (LbL) methodology. A cationic polymer (polyallylamine) and three different anionic polymers with varying absorbance in the UV range (poly(vinyl sulfate), poly(4-styrenesulfonic acid), and humic acid) were used to encapsulate Escherichia coli cells with two different green fluorescent protein (GFP) expression systems: constitutive expression of a UV-excitable GFP (GFPuv) and regulated expression of the intensely fluorescent GFP from amphioxus (GFPa1) through a theophylline-inducible riboswitch. Riboswitches activate protein expression after specific ligand-RNA binding events. Hence, they operate as a cellular biosensor that will activate reporter protein synthesis after exposure to a ligand target. E. coli cells coated with UV-absorbing polymers demonstrated enhanced protection of GFP stability, metabolic activity, and viability after prolonged exposure to radiation from a germicidal lamp. The results show the effectiveness of LbL coatings to provide UV protection to living cells for biotechnological applications.

Riboswitch-based sensor in low optical background

Riboswitches are a type of natural genetic control element that use untranslated sequence in the RNA to recognize and bind to small molecules that regulate expression of that gene. Creation of synthetic riboswitches to novel ligands depends on the ability to screen for analyte binding sensitivity and specificity. In our work, we have coupled a synthetic riboswitch to an optical reporter assay based on fluorescence resonance energy transfer (FRET) between two genetically-coded fluorescent proteins. Specifically, a theophylline-sensitive riboswitch was placed upstream of the Tobacco Etch Virus (TEV) protease coding sequence, and a FRET-based construct, BFP-eGFP or eGFP-REACh, was linked by a peptide encoding the recognition sequence for TEV protease. Cells expressing the riboswitch showed a marked optical difference in fluorescence emission in the presence of theophylline. However, the BFP-eGFP FRET pair posses significant optical background that reduces the sensitivity of a FRET-based assay. To improve the optical assay, we designed a nonfluorescent yellow fluorescent protein (YFP) mutant called REACh (for Resonance Energy-Accepting Chromoprotein) as the FRET acceptor for eGFP. The advantage of using an eGFP-REACh pair is the elimination of acceptor fluorescence which leads to an improved detection of FRET via better signal-to-noise ratio. The EGFP-REACh fusion protein was constructed with the TEV protease cleavage site; thus upon TEV translation, cleavage occurs diminishing REACh quenching and increasing eGFP emission resulting in a 4.5-fold improvement in assay sensitivity.

Screening and selection of artificial riboswitches

Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.