Microfluidic Screening of Electric Fields for Electroporation

Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX[superscript ®], which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX[superscript ®] after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes.United States. Defense Advanced Research Projects Agency (Grant D13AP00025

Microfluidic cell culture chambers with nanoporous walls for chemical communication

Thesis: S.M., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2013.Cataloged from PDF version of thesis.Includes bibliographical references (pages 63-68).Reconstruction of phylogenetic trees based on 16S rRNA gene sequencing reveals that so far only a tiny fraction of microbial diversity has been cultured in the laboratory. One major reason behind this "unculturability" is that many microbes function in symbiosis, frequently exchanging metabolites to sustain their own metabolism, while key exchanged metabolites have hardly been identified. To advance the culturability of diverse microbes we propose a method to engineer a microfluidic co-culture platform, the Microfluidic Cell Culture Chambers, which mimics natural conditions for bacterial growth. The key innovation is to physically isolate bacteria while allowing chemical communication through metabolite diffusion. In this work, the device enables both high-throughput screening and real-time observation of bacterial growth. In our method, we use a porous material, poly(2-hydroxyethyl methacrylateco-ethylene dimethacrylate) (HEMA-EDMA), to fabricate a microwell array with 105 individual culture chambers. Pore size of HEMA-EDMA was confirmed by ESEM imaging to be less than 200 nm, adequate for isolating all identified bacteria. We have video-recorded fluorescence labeled Escherichia coli swimming in confined HEMA-EDMA wells and observed that E. coli is unable to travel between culture chambers. Single-strain E. coli is cultured with the device to test biocompatibility of the device. Syntrophic pairs of E. coli were constructed to test the devices' ability to culture inter-dependent species with physical isolation. In future work, culture of quorum sensing strains is suggested to look into inter-species chemical communication in the Microfluidic Cell Culture Chambers. The future device may be applied to recover uncultured microbial species from natural habitat.by Zhifei Ge.S.M

A compact structured light based otoscope for three dimensional imaging of the tympanic membrane

Three dimensional (3D) imaging of the tympanic membrane (TM) has been carried out using a traditional otoscope equipped with a high-definition webcam, a portable projector and a telecentric optical system. The device allows us to project fringe patterns on the TM and the magnified image is processed using phase shifting algorithms to arrive at a 3D description of the TM. Obtaining a 3D image of the TM can aid in the diagnosis of ear infections such as otitis media with effusion, which is essentially fluid build-up in the middle ear. The high resolution of this device makes it possible examine a computer generated 3D profile for abnormalities in the shape of the eardrum. This adds an additional dimension to the image that can be obtained from a traditional otoscope by allowing visualization of the TM from different perspectives. In this paper, we present the design and construction of this device and details of the imaging processing for recovering the 3D profile of the subject under test. The design of the otoscope is similar to that of the traditional device making it ergonomically compatible and easy to adopt in clinical practice

Bioaerosol generation by raindrops on soil

Aerosolized microorganisms may play an important role in climate change, disease transmission, water and soil contaminants, and geographic migration of microbes. While it is known that bioaerosols are generated when bubbles break on the surface of water containing microbes, it is largely unclear how viable soil-based microbes are transferred to the atmosphere. Here we report a previously unknown mechanism by which rain disperses soil bacteria into the air. Bubbles, tens of micrometres in size, formed inside the raindrops disperse micro-droplets containing soil bacteria during raindrop impingement. A single raindrop can transfer 0.01% of bacteria on the soil surface and the bacteria can survive more than one hour after the aerosol generation process. This work further reveals that bacteria transfer by rain is highly dependent on the regional soil profile and climate conditions

Microbial instrumentation utilizing microfluidics

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Mechanical Engineering, 2017.Cataloged from PDF version of thesis.Includes bibliographical references (pages 126-150).Reconstruction of phylogenetic trees based on 16S rRNA gene sequencing reveals abundant microbial diversity in nature. However, studies of microbiology have been limited by the capabilities to replicate the natural environment or artificially manipulate cells. Advances in microbial instrumentation with microfluidics can break through these challenges. In nature, bacteria live in communities with abundant inter-species chemical communication. To replicate such environments in laboratory conditions, nanoporous microscale microfluidic incubators (NMMIs) for co-culture of multiple species have been developed. The NMMIs enable high-throughput screening and real-time observation of multiple species co-cultured simultaneously. The key innovation in the NMMIs is that they facilitate inter-species communication while maintaining physical isolation between species. NMMIs are a useful tool for the discovery of previously uncultivated organisms and for the study of inter-species microbial interactions. The land and seas are teeming with microbes but one region of the environment often neglected is the air. Large numbers of microbes are present in air yet little is known about the mechanisms that lead to their dispersion. We have elucidated one such dispersion mechanisms involving rain and soil bacteria. The experimental system replicates the process of raindrops impinging on soil surfaces that contain bacteria. It is demonstrated that up to 0.01% of soil bacteria can be dispersed by aerosolization and survive for more than an hour after the aerosolization process. This mechanism can be relevant for the investigation of climate change, pathogenic disease transmission, and geographic migration of bacteria. In spite of the challenges outlined above there are thousands of known species of bacteria that have been catalogued and genetically sequenced. However, few of these organisms are amenable to modem genetic manipulation tools. Thus there is a great benefit for a tool that accelerates the development of efficient genetic transformation protocols. We have developed a microfluidic electroporation device to address this challenge. The key novelty is the microchannel geometry which applies a linear electric field gradient to each sample. This design enables rapid determination of the electric field that leads to quantifiable bacterial electroporation. Bacterial strains with both industrial and medical relevance have been successfully characterized using this assay.by Zhifei Ge.Ph. D

PBQ-Enhanced QUIC: QUIC with Deep Reinforcement Learning Congestion Control Mechanism

Currently, the most widely used protocol for the transportation layer of computer networks for reliable transportation is the Transmission Control Protocol (TCP). However, TCP has some problems such as high handshake delay, head-of-line (HOL) blocking, and so on. To solve these problems, Google proposed the Quick User Datagram Protocol Internet Connection (QUIC) protocol, which supports 0-1 round-trip time (RTT) handshake, a congestion control algorithm configuration in user mode. So far, the QUIC protocol has been integrated with traditional congestion control algorithms, which are not efficient in numerous scenarios. To solve this problem, we propose an efficient congestion control mechanism on the basis of deep reinforcement learning (DRL), i.e., proximal bandwidth-delay quick optimization (PBQ) for QUIC, which combines traditional bottleneck bandwidth and round-trip propagation time (BBR) with proximal policy optimization (PPO). In PBQ, the PPO agent outputs the congestion window (CWnd) and improves itself according to network state, and the BBR specifies the pacing rate of the client. Then, we apply the presented PBQ to QUIC and form a new version of QUIC, i.e., PBQ-enhanced QUIC. The experimental results show that the proposed PBQ-enhanced QUIC achieves much better performance in both throughput and RTT than existing popular versions of QUIC, such as QUIC with Cubic and QUIC with BBR

PBQ-Enhanced QUIC: QUIC with Deep Reinforcement Learning Congestion Control Mechanism

Currently, the most widely used protocol for the transportation layer of computer networks for reliable transportation is the Transmission Control Protocol (TCP). However, TCP has some problems such as high handshake delay, head-of-line (HOL) blocking, and so on. To solve these problems, Google proposed the Quick User Datagram Protocol Internet Connection (QUIC) protocol, which supports 0-1 round-trip time (RTT) handshake, a congestion control algorithm configuration in user mode. So far, the QUIC protocol has been integrated with traditional congestion control algorithms, which are not efficient in numerous scenarios. To solve this problem, we propose an efficient congestion control mechanism on the basis of deep reinforcement learning (DRL), i.e., proximal bandwidth-delay quick optimization (PBQ) for QUIC, which combines traditional bottleneck bandwidth and round-trip propagation time (BBR) with proximal policy optimization (PPO). In PBQ, the PPO agent outputs the congestion window (CWnd) and improves itself according to network state, and the BBR specifies the pacing rate of the client. Then, we apply the presented PBQ to QUIC and form a new version of QUIC, i.e., PBQ-enhanced QUIC. The experimental results show that the proposed PBQ-enhanced QUIC achieves much better performance in both throughput and RTT than existing popular versions of QUIC, such as QUIC with Cubic and QUIC with BBR

Uplink Throughput Maximization in UAV-Aided Mobile Networks: A DQN-Based Trajectory Planning Method

This paper focuses on the unmanned aerial vehicles (UAVs)-aided mobile networks, where multiple ground mobile users (GMUs) desire to upload data to a UAV. In order to maximize the total amount of data that can be uploaded, we formulate an optimization problem to maximize the uplink throughput by optimizing the UAV’s trajectory, under the constraints of the available energy of the UAV and the quality of service (QoS) of GMUs. To solve the non-convex problem, we propose a deep Q-network (DQN)-based method, in which we employ the iterative updating process and the Experience Relay (ER) method to reduce the negative effects sequence correlation on the training results, and the ε-greedy method is applied to balance the exploration and exploitation, for achieving the better estimations of the environment and also taking better actions. Different from previous works, the mobility of the GMUs is taken into account in this work, which is more general and closer to practice. Simulation results show that the proposed DQN-based method outperforms a traditional Q-Learning-based one in terms of both convergence and network throughput. Moreover, the larger battery capacity the UAV has, the higher uplink throughput can be achieved

Research on Improving the Working Efficiency of Hydraulic Jet Submarine Cable Laying Machine

The anchoring and hooking of ships, bedrock friction and biological corrosion threaten the safety and stability of submarine cables. A hydraulic jet submarine cable laying machine manages to bury the submarine cables deep into the seabed, and effectively reduces the occurrence of external damage to the submarine cables. This machine uses a hydraulic jet system to realize trenching on the seabed. However, the hydraulic jet submarine cable laying machine has complicated operation and high power consumption with high requirements on the mother ship, and it is not yet the mainstream trenching method. In this paper, a mathematical model for the hydraulic jet nozzle of the submarine cable laying machine is established, and parameters that affect the trenching efficiency are studied. The effects of jet target distance, flow, angle and nozzle spacing on the working efficiency of the burying machine are analyzed by setting up a double-nozzle model. The results of the theory, numerical simulation and experiment show that the operational efficiency of the hydraulic jet submarine cable laying machine can be distinctly improved by setting proper jet conditions and parameters