Extended cell-free protein expression system for amino acid labeling and structural biology at Miami University's Center for Bioinformatics and Functional Genomics (CBFG)

Project Title: Extended cell-free protein expression system for amino acid labeling and structural biology studies at Miami University's Center for Bioinformatics and Functional Genomics (CBFG).

Project Lead: Carole Dabney-Smith

Email: smithac5@miamioh.edu

Phone: (513) 529-8091

Affiliation: CAS

Other Team Members: Andor Kiss, Gary A. Lorigan

Project Details: This project is a request for a temperature regulated, heating and cooling capable, mixing, dry incubator with accessories capable of handling tube with different volumes and a starter expression kit. This will enable undergraduate and graduate student users to produce microgram to milligram quantities of in vitro expressed protein in a semi-high throughput fashion to aid in the investigation of protein structure/function relationships of any protein. One consistent barrier that impedes the progress of, discovery of structure/function relationships has been the ability to reliably and quickly generate protein, e.g., with single amino acid changes throughout the protein to understand the structure of that region of the protein or the catalytic activity of an enzyme. The addition of equipment to the CBFG that enhance the ability to quickly generate many proteins at the same time allows student researchers to more quickly survey a protein by amino acid changes or an array of different proteins to identify the candidates needing further study. The CBFG currently does not have a dry incubator system with temperature control and mixing features nor other capabilities of cell free expression. This would introduce new capabilities to the CBFG Core Protein Facility enabling decreased screening times necessary to pinpoint critical amino acids, particularly for protein structural determination. The Eppendorf ThermoMixer C and a starter expression kit are the best tools for this purpose because of their adaptability and scalability due to the many different interchangeable blocks and reagents supplied. This system would allow users to start on a small scale for initial screening and then scale up for more in depth analysis of a particular protein. We are asking for two such units so that multiple investigators can use one unit routinely for small scale screening and the other unit for larger scale processes. Our current methods to make different proteins involve overexpression in bacterial cells and are time and space intensive because the cells take time (3-4 days from start to finish) and space to grow (floor space in labs). The extended cell-free expression system takes advantage of purified protein expression components eliminating the need to grow the bacterial cells and allowing investigators to express many different proteins at once (hours) in a small footprint (on the benchtop). We are therefore requesting $19,088 for the acquisition of two (2x) units the Eppendorf Thermomixer C plus block attachments as well as a starter expression kit from Cell Free Sciences to enable us to optimise and generate institutional knowledge in the area of protein expression (please see attached quotes). These instruments do not require a service contract for yearly maintenance, making this is a one-time acquisition request.

Problem Project Attempts to Solve: The problem that we are facing is the ability to efficiently generate purified and labeled proteins in a high throughput manner, ensuring that the downstream structure/function analysis is as efficient as possible. While structure/function studies are not entirely empirical in practice most strategies are somewhat empirical, which means an investigator (student or otherwise) makes a single amino acid change in a protein of interest, has to do the molecular biology methods to introduce the gene for that protein into bacterial cells for overexpression and then purify the protein. This is a time consuming process that may or may not result in expression of the protein. A good solution would be to have the capabilities to do extended cell-free protein expression. This would permit students (undergraduate biochemistry or biotechnology lab classes, undergraduate researchers and graduate students) to express proteins efficiently with high throughput while overcoming the potential downside of difficult protein expression in bacterial cells. It would also permit the addition of new lab exercises that take advantage of the screening expression of protein variants to demonstrate the effect amino acids changes have on enzymatic activity.

Does this project focus on Graduate Studies?: Yes

If yes, explain: This request will benefit undergraduate and graduate student protein science researchers. The benefit would be divided in approximately a %60 / % 40 split between graduate students and undergraduates. This is due to all the PIs involved in this project are strongly committed to training of undergraduate researchers as well as providing an opportunity for their graduate students to gain mentoring experience during their graduate careers. These dual research objectives will be accomplished in two key ways: (1) by increasing the efficiency with which proteins with interesting amino acid changes are discovered, thus providing more in depth analysis of protein structure/function relationships; and (2) through training to use state of the art methods providing students the best training we can to help them be competitive for jobs after graduation - both at the post-doctoral and the post-graduate levels.

Does it meet tech fee criteria?: High throughput extended cell-free protein expression is the next generation for quickly expressing, labeling, purifying, and testing proteins for activity or for structural studies. These modern biochemistry and molecular biology skill sets are extremely desirable for both undergraduate students applying for graduate and/or professional (medical) schools or for those seeking immediate employment after graduation. For graduate students and even post-doctoral fellows, these are skills that greatly benefit their future employment prospects in biochemistry or biophysical chemistry fields. Therefore, in order for Miami University to remain competitive in recruitment of undergraduate students in the biological and biochemical sciences, especially the pre-health focused students (in which high throughput extended cell-free protein expression is being used to generate protein for structural studies in hospital research laboratories), we need to have a complete pipeline for training purposes. The innovation of this project is to enable our students (undergraduate and graduate) to be trained in the latest, most innovative approaches to science and technology thereby enhancing their research experiences individually as well as collaboratively, e.g., through the DUOS Program. By gaining familiarity with this technology (extended cell-free protein expression), students will be able to develop new assays and new approaches. Not having a complete preparation pipeline is a present a hurdle to the students understanding of the process and to our ability to produce graduates of the highest caliber.

How will you assess the project?: An expected outcome of the acquisition of the Eppendorf ThermoMixer C units with the accessory blocks and a starter expression kit will be the cell free overexpression of many different proteins at once for concomitant structure/function studies. This would enhance research involving undergraduate students and graduate students individually and through collaboration through the DUOS Program. Currently this option is rarely used at Miami due to the lack of equipment to make high throughput feasible. We also intend to implement the cell free expressions system into the CHM438 Biochemistry Lab to introduce modern technologies to students. Assessment of the value to students will be by collecting metric data on peer-reviewed papers published, podium and poster presentations, and completed MS theses and PhD dissertations.

Have you received tech fee funding in the past?: No

What happens to this project in year two?: This project request is for an acquisition of the instrumentation. If awarded, the two ThermoMixer C units with accessory blocks will be housed and maintained at the CBFG. The CBFG has greater than a decade long history of successful maintaining and training users (undergraduates, graduates, post-docs, technicians, visiting scientists, and PIs) on the proper usage of its instrumentation to achieve optimal results. With two permanent, full-time staff (Dr. Andor Kiss (Supervisor) and Ms. Xiaoyun Deng (Senior Technician) as well as a Faculty Director (Prof Luis Actis). There is no service contract required and any ongoing costs such as consumables will be at the expense of the individual faculty. In the event that the instruments break, the CBFG has enough operational resources to repair the instrument.

Hardware: Eppendorf ThermoMixer C plus accessories and Cell Free Sciences starter kits $ 19087.939999999999

Total Budget: $19087.939999999999