The Sinskey Lab is collaborating closely with the Chemical Engineering department and the Sloan school of management at MIT, as well as various Chinese partners, to mitigate the risk of economically motivated adulteration in Chinese food supply chains. Specifically, we are working with the Strano group to develop a multiplexed contaminant detection platform that will be able to detect pathogens, heavy metals, and small molecules using one device. As part of this effort, we are developing single-domain antibodies (sdAb) from camelids that can specifically recognize these various adulterants and couple them with infrared optical sensors for rapid detection. Our goal is to replace current qualitative detection kits that can only detect one adulterant with a rapid qualitative platform that can detect multiple contaminants in one sample.
The Sinskey Lab is collaborating with the Center for Biomedical Innovation (CBI) at MIT to develop a bioassay that could fundamentally transform approaches to unknown toxicant identification by measuring the biological effects of tainted foods rather than testing for one or a few likely toxicants at a time. Our goal is to 1) develop an extraction method from jerky treat products, 2) develop a bioassay capable of monitoring over 500 different phenotypic signatures from cells exposed to these extracts, 3) create a library of phenotypic signatures for suspected adulterants, 4) and accurately prove that we could predict jerky treat toxicity from case study samples.
Over the past 60 years, C. glutamicum has been well known as an excellent producer of ʟ-amino acids. Recent advances in metabolic and synthetic biology have expanded the portfolio of chemicals that can be produced from C. glutamicum, but it is still difficult to synthesize these compounds at an industrially relevant scale. Due to the increasing worldwide demand of amino acids and other novel compounds, there is a pressing need for rapid improvement in strain engineering and production yields of chemical compounds in C. glutamicum and other production strains. Current technology for strain engineering and genome modification in C. glutamicum is notoriously slow. Gene clusters are deleted by a double-crossover homologous recombination event using the suicide plasmid pK18mobsacB. This process takes at least a week if successful successive mutations have to be introduced sequentially, which substantially increases the time needed to edit the genome. In order to improve genome editing and transformation rates, the Sinskey lab is developing a recombinase-based genome engineering toolset that will allow for the quick introduction of mutations with high frequency.
The Sinskey lab has recently began a new area of research looking at enzymatic recycling of common plastics, such as PET. Our interests include improving enzyme activity using both in vitro and in silico directed evolution as well as using rational design. In collaboration with the Foundry at the Broad, we are designing an optimized strategy for the rapid screening of enhanced enzymatic activity that can be implemented for the directed evolution of such enzymes.
The Sinskey Lab is focused on engineering Corynebacterium glutamicum to produce high value compounds, such as shikimate and its many high-value aromatic derivatives. For example, shikimate has three chrial centers that makes it difficult to chemically synthesize, forcing most current shikimate production to come from plant extraction. This procedure, however, is costly, deterimental to the environment, and yields low amounts. In order to produce shikimate and its derivatives in a sustainable manner, we are engineering new pathways into C. glutamicum using synthetic biological circuits to reduce starting cost and improve yields. In addition, the Sinskey lab is focused on engineering Corynebacterium glutamicum to produce carotenoids, such as α-carotene. Commercially, α-carotene is used as a food colorant, animal feed supplement, cosmetic, and a nutraceutical due to its ability to reduce the risk of cardiovascular disease and cancer. Most current α-carotene production, however, comes from plant extraction, which is both costly and results in low yields. Our goal is to use genetic engineering tools and pathway engineering to produce and accumulate α-carotene in C. glutamicum. This will allow for a more sustainable fermentation process that will eliminate the need for expensive plant extraction and improve overall yields.
The Sinskey lab is focused on using novel strategies to engineer fatty acid production in Corynebacterium glutamicum, with special interest in the production of medium chain fatty acids (MCFA). MCFA are fatty acids with a chain length between C6-C12 that can be found in seeds and products like coconut oil, palm kernel oil and coconut milk. They are also known as precursors for many chemicals and industrial biofuels. Since there are still no available industrial microbes with known pathways that can produce MCFA, our goal is to use genetically engineering tools to introduce MCFA production in C. glutamicum. To achieve this goal, we are identifying genes from oil palms and other sources that are involved in the biosynthesis of MCFAs and introducing them into C. glutamicum.
The Sinskey lab is leading an effort to reduce the genome of C. glutamicum to reduce complexity, improve overall fitness, increase genomic stability, and reduce the time required to produce production strains. We are working closely with the Center for Biotechnology (CiBeTec) at the University of Bielefeld and the National University of Singapore (NUS) to identify essential genes that are required for growth and eliminate genes that do not affect overall fitness. This new strain will be validated by comparing both the yield and time needed to engineer the production of high value compounds with wild-type strains.
Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible polymers synthesized by a large number of microorganisms. Under nutrient limitation, some bacteria are able to accumulate large amounts of PHAs as carbon and energy reservoirs. The Sinskey lab is focused on generating strategies to use PHAs as micro- and nano-bioreactors for the biosynthesis of hydrophobic compounds such as steroids.
A project collaborated at the Center for Biomedical Innovation (CBI), chemical engineering and biology, all at MIT, aims to develop an insightful production process for recombinant adeno-associated virus (AAV). AAV has gained increasing importance in the era of gene and cell therapies that have the potential to cure challenging diseases. Currently, however, the complex biology in the recombinant production for AAV using mammalian cells still limits the yield and quality of gene therapy. This project is aimed to tackle the challenge by achieving (1) continuous operation of AAV production; (2) integration of online monitoring technology; (3) employment of mechanistic models that elucidate the kinetics in the biological system and (4) an enhancement of process optimization and control by bringing all together.
Continuous bioprocessing is an important initiative in the biopharmaceutical landscape, allowing higher yields of therapeutic proteins for lower processing costs and enabling real-time control for the process itself. In a collaboration between MIT’s Center for Biomedical Innovation, the Chemical Engineering department, the Biology department, and the Sloan School of Management, we aim to leverage first-principles and data-driven modeling strategies to understand and control the impact of continuous monoclonal antibody manufacture on the ultimate product quality. We are designing and assembling a lab-scale continuous bioprocessing testbed to assist in development of plant wide models and to allow determination of the risk associated with application of non-ideal process models.
Novel T-cell-based (CAR-T) therapies cure previously untreatable blood cancers and the bio-manufactured T-cells should be free from adventitious agents introduced during the cell manufacturing processes. The Sinskey Lab collaborates with the Center for Biomedical Innovation (CBI) at MIT to develop a method to ensure rapid, sensitive, and untargeted detection of adventitious agents during cell bio-manufacturing using third-generation sequencing approaches. Our study aims to improve cell manufacturing sterility testing by developing rapid methods for sensitive and accurate detection of viruses using metagenomic sequencing.
The project in the Sinskey Lab collaborated with Center for Biomedical Innovation (CBI) at MIT aims to design, develop, and manufacture a virus-like particle (VLP) vaccine for SARS-CoV-2. We are generating a VLP expression system in a HEK 293 host that efficiently creates a consistent VLP product. This production platform was designed such that the Spike protein characteristic of SARS-CoV-2 can be easily modified, allowing the vaccine to be modified in response to critical viral mutations. The resulting VLP production system will be scaled up and used for advanced process development. Advanced process analytical technology (PAT) is applied to fully characterize the process and product, establishing a baseline Critical Quality Attribute (CQA) profile to ensure a consistently safe and effective product. Data collected during process development is used to generate advanced processing models and robust control strategies for optimal production of the VLP vaccine. A continuous manufacturing mode can be realized by integrating perfusion bioreactor and downstream process. The techniques developed throughout the work can be applied to accelerate the vaccine pipeline in response to future pandemics.