Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Programming Biomaterial Interactions Using Engineered Living Cells.

We have developed a biomaterials interface that allows the properties of a functionalized surface to be controlled by a population of genetically engineered bacteria. This interface was engineered by linking a genetically modified E. coli strain with a chemically functionalized surface. Critically, the E. coli was engineered to upregulate the production of biotin when induced by a small signaling molecule. This biotin would then interact with the functionalized surface to modulate the surface's binding dynamics. In this chapter, we detail three protocols: one protocol for developing a population of biotin-producing genetically engineered cells, and two protocols for creating different types of functionalized surfaces. These methods will enable scientists to readily explore strategies for controlling surface-based material assembly and modification using a linked culture of engineered cells.

Engineering a living biomaterial via bacterial surface capture of environmental molecules.

Synthetic biology holds significant potential in biomaterials science as synthetically engineered cells can produce new biomaterials, or alternately, can function as living components of new biomaterials. Here, we describe the creation of a new biomaterial that incorporates living bacterial constituents that interact with their environment using engineered surface display. We first developed a gene construct that enabled simultaneous expression of cytosolic mCherry and a surface-displayed, catalytically active enzyme capable of covalently bonding with benzylguanine (BG) groups. We then created a functional living material within a microfluidic channel using these genetically engineered cells. The material forms when engineered cells covalently bond to ambient BG-modified molecules upon induction. Given the wide range of materials amenable to functionalization with BG-groups, our system provides a proof-of-concept for the sequestration and assembly of BG-functionalized molecules on a fluid-swept, living biomaterial surface.

Using Synthetic Biology to Engineer Living Cells That Interface with Programmable Materials.

We have developed an abiotic-biotic interface that allows engineered cells to control the material properties of a functionalized surface. This system is made by creating two modules: a synthetically engineered strain of E. coli cells and a functionalized material interface. Within this paper, we detail a protocol for genetically engineering selected behaviors within a strain of E. coli using molecular cloning strategies. Once developed, this strain produces elevated levels of biotin when exposed to a chemical inducer. Additionally, we detail protocols for creating two different functionalized surfaces, each of which is able to respond to cell-synthesized biotin. Taken together, we present a methodology for creating a linked, abiotic-biotic system that allows engineered cells to control material composition and assembly on nonliving substrates.

Low-cost feedback-controlled syringe pressure pumps for microfluidics applications.

Microfluidics are widely used in research ranging from bioengineering and biomedical disciplines to chemistry and nanotechnology. As such, there are a large number of options for the devices used to drive and control flow through microfluidic channels. Commercially available syringe pumps are probably the most commonly used instruments for this purpose, but are relatively high-cost and have inherent limitations due to their flow profiles when they are run open-loop. Here, we present a low-cost ($110) syringe pressure pump that uses feedback control to regulate the pressure into microfluidic chips. Using an open-source microcontroller board (Arduino), we demonstrate an easily operated and programmable syringe pump that can be run using either a PID or bang-bang control method. Through feedback control of the pressure at the inlets of two microfluidic geometries, we have shown stability of our device to within ±1% of the set point using a PID control method and within ±5% of the set point using a bang-bang control method with response times of less than 1 second. This device offers a low-cost option to drive and control well-regulated pressure-driven flow through microfluidic chips.

Programming Surface Chemistry with Engineered Cells.

We have developed synthetic gene networks that enable engineered cells to selectively program surface chemistry. E. coli were engineered to upregulate biotin synthase, and therefore biotin synthesis, upon biochemical induction. Additionally, two different functionalized surfaces were developed that utilized binding between biotin and streptavidin to regulate enzyme assembly on programmable surfaces. When combined, the interactions between engineered cells and surfaces demonstrated that synthetic biology can be used to engineer cells that selectively control and modify molecular assembly by exploiting surface chemistry. Our system is highly modular and has the potential to influence fields ranging from tissue engineering to drug development and delivery.

A Model of a Synthetic Biological Communication Interface between Mammalian Cells and Mechatronic Systems.

The creation of communication interfaces between abiotic and biotic systems represents a significant research challenge. In this work, we design and model a system linking the biochemical signaling pathways of mammalian cells to the actions of a mobile robotic prosthesis. We envision this system as a robotic platform carrying an optically monitored bioreactor that harbors mammalian cells. The cellular, optical signal is captured by an onboard fluorescent microscope and converted into an electronic signal. We first present a design for the overall cell-robot system, with a specific focus on the design of the synthetic gene networks needed for the system. We use these synthetic networks to encode motion commands within the cell's endogenous, oscillatory calcium signaling pathways. We then describe a potential system whereby this oscillatory signal could be outputted and monitored as a change in cellular fluorescence. Next, we use the changes resulting from the synthetic biological modifications as new parameters in a simulation of a well-established mathematical model for intracellular calcium signaling. The resulting signal is processed in the frequency domain, with specific frequencies activating cognate robot motion subroutines.

Exploring Host-Microbiome Interactions using an in Silico Model of Biomimetic Robots and Engineered Living Cells.

The microbiome's underlying dynamics play an important role in regulating the behavior and health of its host. In order to explore the details of these interactions, we created an in silico model of a living microbiome, engineered with synthetic biology, that interfaces with a biomimetic, robotic host. By analytically modeling and computationally simulating engineered gene networks in these commensal communities, we reproduced complex behaviors in the host. We observed that robot movements depended upon programmed biochemical network dynamics within the microbiome. These results illustrate the model's potential utility as a tool for exploring inter-kingdom ecological relationships. These systems could impact fields ranging from synthetic biology and ecology to biophysics and medicine.