Fluigi: an end-to-end software workflow for microfluidic design

One goal of synthetic biology is to design and build genetic circuits in living cells for a range of applications with implications in health, materials, and sensing. Computational design methodologies allow for increased performance and reliability of these circuits. Major challenges that remain include increasing the scalability and robustness of engineered biological systems and streamlining and automating the synthetic biology workflow of “specify-design-build-test.” I summarize the advances in microfluidic technology, particularly microfluidic large scale integration, that can be used to address the challenges facing each step of the synthetic biology workflow for genetic circuits. Microfluidic technologies allow precise control over the flow of biological content within microscale devices, and thus may provide more reliable and scalable construction of synthetic biological systems. However, adoption of microfluidics for synthetic biology has been slow due to the expert knowledge and equipment needed to fabricate and control devices. I present an end-to-end workflow for a computer-aided-design (CAD) tool, Fluigi, for designing microfluidic devices and for integrating biological Boolean genetic circuits with microfluidics. The workflow starts with a ``netlist" input describing the connectivity of microfluidic device to be designed, and proceeds through placement, routing, and design rule checking in a process analogous to electronic computer aided design (CAD). The output is an image of the device for printing as a mask for photolithography or for computer numerical control (CNC) machining. I also introduced a second workflow to allocate biological circuits to microfluidic devices and to generate the valve control scheme to enable biological computation on the device. I used the CAD workflow to generate 15 designs including gradient generators, rotary pumps, and devices for housing biological circuits. I fabricated two designs, a gradient generator with CNC machining and a device for computing a biological XOR function with multilayer soft lithography, and verified their functions with dye. My efforts here show a first end-to-end demonstration of an extensible and foundational microfluidic CAD tool from design concept to fabricated device. This work provides a platform that when completed will automatically synthesize high level functional and performance specifications into fully realized microfluidic hardware, control software, and synthetic biological wetware

Design and characterization of artificial transcriptional terminators

Thesis (M. Eng.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2007.Includes bibliographical references (p. 103-104).Design and characterization of artificial transcriptional terminators. Ten new terminators were designed based on previous research of terminator structure and termination efficiency. The terminators were built by PCR extension, ligated into a BioBrick plasmid backbone, and transformed into TOP10 cells. Characterization devices were built to test the terminators. Input and output of the terminator were measured by expression of RFP and GFP. Charaterization devices were then placed into the E. coli strain CW2553/pJAT18, which hijacks the arabinose transport system to provide controlled input to the terminator. Of the ten terminators designed and tested, BBa_B1002, BBa_B1004, BBa_B1006 and BBa_B1010 proved to be strong terminators with termination efficiencies above 90%. These terminators may be obtained from the Registry of Standarized Parts at parts.mit.edu.by Haiyao Huang.M.Eng

MINT - Microfluidic Netlist

Fluigi is a microfluidic design framework that allows researchers to realize abstract descriptions of liquid flow relationships automatically as physical devices and corresponding control software. Its goal is to provide synthetic biology researchers with the tools to use microfluidics for novel computation, discovery, and test applications. A critical component of this work-flow is MINT, a format for describing the microfluidic components and the connectivity of the control and flow layers in the microfluidic device. This work describes MINT and where it falls in the larger Fluigi software flow

Unexpected enrichment of thallium and its geochemical behaviors in soils impacted by historically industrial activities using lead‑zinc carbonate minerals

: Thallium is a trace metal with severe toxicity. Contamination of thallium (Tl) generated by steel and non-ferrous metals industry is gaining growing concern worldwide. However, little is known on Tl contamination owing to industrial activities using carbonate minerals. This study revealed abundant geochemical mobile/bioavailable Tl (> 65.7%, in average; mostly in oxidizable fraction) in soils from a carbonate-hosted PbZn ore utilizing area in China for the first time. Unexpected Tl enrichment was observed in soil accompanying with 3655, 7820, 100.1, 27.3 and 29.9 mg/kg (in average) of Pb, Zn, As, Cd and Sb, respectively. Characterization using X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) analysis further confirmed that historical industrial activities impose anthropogenic catastrophic effects on the local agricultural soil system. The ecological and health risk assessment of heavy metal(loid)s in soils proclaimed serious potential non-carcinogenic risks of Pb and V to adults, and Pb, Tl and As to children. Sequential extraction analysis showed that Tl, as well as Pb, Zn, Mn, Co, and Cd, mainly existed in the mobile fractions (exchangeable/acid-extractable, reducible and oxidizable), indicating an ecological risk of biological accumulation of multiple metal(loid)s in this area. These findings provide a theoretical basis for taking appropriate remediation measures in order to ensure safety of soils in such industrial areas likewise

Targeting ZDHHC9 potentiates anti-programmed death-ligand 1 immunotherapy of pancreatic cancer by modifying the tumor microenvironment

Immune checkpoint blockade (ICB) therapy targeting the programmed death 1/programmed death-ligand 1 (PD-1/PD-L1) axis has achieved considerable success in treating a wide range of cancers. However, most patients with pancreatic cancer remain resistant to ICB. Moreover, there is a lack of optimal biomarkers for the prediction of response to this therapy. Palmitoylation is mediated by a family of 23 S-acyltransferases, termed zinc finger Asp‐His‐His‐Cys-type palmitoyltransferases (ZDHHC), which precisely control various cancer-related protein functions and represent promising drug targets for cancer therapy. Here, we revealed that tumor cell-intrinsic ZDHHC9 was overexpressed in pancreatic cancer tissues and associated with impaired anti-tumor immunity. In syngeneic pancreatic tumor models, the knockdown of ZDHHC9 expression suppressed tumor progression and prolonged survival time of mice by modifying the immunosuppressive (‘cold’) to proinflammatory (‘hot’) tumor microenvironment. Furthermore, ZDHHC9 deficiency sensitized anti-PD-L1 immunotherapy mainly in a CD8+ T cell dependent manner. Lastly, we employed the ZDHHC9-siRNA nanoparticle system to efficiently silence ZDHHC9 in pancreatic tumors. Collectively, our findings indicate that ZDHHC9 overexpression in pancreatic tumors is a mechanism involved in the inhibition of host anti-tumor immunity and highlight the importance of inactivating ZDHHC9 as an effective immunotherapeutic strategy and booster for anti-PD-L1 therapy against pancreatic cancer