Statistical Modeling of β-galactosidase Inhibition During Lactose Hydrolysis

A statistical model approach called response surface methodology was used to describe the product and substrate inhibition effect on β-galactosidase enzyme during lactose hydrolysis. The effect of independent variables, namely the initial concentrations of lactose (73 - 146 mM), galactose (44 - 122 mM) and glucose (83 - 167 mM) on the reaction rate of β-galactosidase was evaluated. The enzymatic reaction rate was influenced by both combined and individual effects of all the substrate and products. Although, glucose acted as an activator at low lactose and low galactose concentrations, glucose caused the inhibition of β-galactosidase at higher concentrations of lactose and galactose. The effect of galactose concentration on β-galactosidase enzyme was in the direction of inhibition. At low lactose concentrations and high glucose concentrations, galactose concentration became more effective on the reaction rate.     

Genetic Logic Gates Enable Patterning of Amyloid Nanofibers

Distinct spatial patterning of naturally produced materials is observed in many cellular structures and even among communities of microorganisms. Reoccurrence of spatially organized materials in all branches of life is clear proof that organization is beneficial for survival. Indeed, organisms can trick the evolutionary process by using organized materials in ways that can help the organism to avoid unexpected conditions. To expand the toolbox for synthesizing patterned living materials, Boolean type “AND” and “OR” control of curli fibers expression is demonstrated using recombinases. Logic gates are designed to activate the production of curli fibers. The gates can be used to record the presence of input molecules and give output as CsgA expression. Two different curli fibers (CsgA and CsgA‐His‐tag) production are then selectively activated to explore distribution of monomers upon coexpression. To keep track of the composition of fibers, CsgA‐His‐tag proteins are labeled with nickel–nitrilotriacetic acid (Ni–NTA‐) conjugated gold nanoparticles. It is observed that an organized living material can be obtained upon inducing the coexpression of different CsgA fibers. It is foreseen that living materials with user‐defined curli composition hold great potential for the development of living materials for many biomedical applications.     

Cellular Biocatalysts Using Synthetic Genetic Circuits for Prolonged and Durable Enzymatic Activity

Cellular biocatalysts hold great promise for the synthesis of difficult to achieve compounds, such as complex active molecules. Whole-cell biocatalysts can be programmed through genetic circuits to be more efficient, but they suffer from low stability. The catalytic activity of whole cells decays under stressful conditions, such as prolonged incubation times or high temperatures. In nature, microbial communities cope with these conditions by forming biofilm structures. In this study, it is shown that the use of biofilm structures can enhance the stability of whole-cell biocatalysts. We employed two different strategies to increase the stability of whole-cell catalysts and decrease their susceptibility to high temperature. In the first approach, the formation of a biofilm structure is induced by controlling the expression of one of the curli component, CsgA. The alkaline phosphatase (ALP) enzyme was used to monitor the catalytic activity of cells in the biofilm structure. In the second approach, the ALP enzyme was fused to the CsgA curli fiber subunit to utilize the protective properties of the biofilm on enzyme biofilms. Furthermore, an AND logic gate is introduced between the expression of CsgA and ALP by toehold RNA switches and recombinases to enable logical programming of the whole-cell catalyst for biofilm formation and catalytic action with different tools. The study presents viable approaches to engineer a platform for biocatalysis processes.     

Peptide-mediated constructs of quantum dot nanocomposites for enzymatic control of nonradiative energy transfer

A bottom-up approach for constructing colloidal semiconductor quantum dot (QDot) nanocomposites that facilitate nonradiative Fo?rster-type resonance energy transfer (FRET) using polyelectrolyte peptides was proposed and realized. The electrostatic interaction of these polypeptides with altering chain lengths was probed for thermodynamic, structural, and morphological aspects. The resulting nanocomposite film was successfully cut with the protease by digesting the biomimetic peptide layer upon which the QDot assembly was constructed. The ability to control photoluminescence decay lifetime was demonstrated by proteolytic enzyme activity, opening up new possibilities for biosensor applications.     

Statistical Modeling of β-galactosidase Inhibition During Lactose Hydrolysis

A statistical model approach called response surface methodology was used to describe the product and substrate inhibition effect on β-galactosidase enzyme during lactose hydrolysis. The effect of independent variables, namely the initial concentrations of lactose (73 – 146 mM), galactose (44 – 122 mM) and glucose (83 – 167 mM) on the reaction rate of β-galactosidase was evaluated. The enzymatic reaction rate was influenced by both combined and individual effects of all the substrate and products. Although, glucose acted as an activator at low lactose and low galactose concentrations, glucose caused the inhibition of β-galactosidase at higher concentrations of lactose and galactose. The effect of galactose concentration on β-galactosidase enzyme was in the direction of inhibition. At low lactose concentrations and high glucose concentrations, galactose concentration became more effective on the reaction rate.