ChBE Summer Scholars, Katie Rines and Nick Horelik, work on summer research Grant

This summer, students in chemical and biological engineering spent their summers researching topics ranging from engineering better ways for the body to fight off disease to developing technology that might help reduce greenhouse gases. ChBE students Katie Rines ('08) and Nick Horelik ('09) are two of many engineering students expanding their knowledge as part of the Tufts Summer Scholars Program. The Tufts Summer Scholars Program is a university-wide initiative that offers research apprenticeships with faculty or clinical mentors to motivated Tufts undergraduates. With a $3,500 scholarship, the program gives students a chance to be on the front line of discovery and scholarship at Tufts.

Learn about immunotherapy from Katie		Find out about nanotechnology with Nick

Name: Katie Rines, ChBE'08
Project title: Cellular Engineering for Therapeutic Applications
Advisor: Assistant Professor Blaine Pfeifer

What it's all about: One of medicine's ultimate goals is to home in on the specifics of a disease without causing unintended consequences in other areas of the body. For example, in treating cancer, chemical agents delivered during chemotherapy can block cell division or destroy rapidly dividing cells. However, as a result, other rapidly dividing cells, like hair cells, can be affected and leave patients bald as a side effect of treatment. By genetically modifying the bacterium E. coli, chemical engineers may be able to stimulate the body's immune system to more specifically respond to diseased cells. E. coli is the workhorse of the biochemical engineering world as its simple and well understood DNA structure can be engineered to include genes that could produce proteins to heighten the body's immune system to disease—a process called immunotherapy. First, however, chemical engineers must ensure that targeted immune cells, when introduced to these bacterial vectors, are actually incorporating the genetic material. Katie Rines uses fluorescent microscopy techniques to ensure this process is happening.

How it works: Katie worked with Saba Parsa, a chemical and biological engineering master's student, to first produce strains of E. coli with appropriate genes to both deliver the bacterial information into the immune cells and then a "dummy" protein to induce an immune response. This dummy protein, green fluorescent protein (GFP), does exactly what its name implies—fluoresces green when expressed. After the bacterial strains were created, Katie combined them with a type of mammalian immune cells called macrophages. During the hour-long co-incubation, the mammalian cells engulf the bacterial cells into a vesicle, called a phagosome. Then, the protein expressed by the genetically modified bacteria, listeriolysin-O (LLO), breaks open the phagosome to dump the genetic information, including the GFP, inside the immune cells. "The LLO facilitates the immunotherapeutic response to the delivered gene or protein," Katie said. The immune cells' nuclei take up the GFP gene and then produce the fluorescent protein. "Those mammalian cells that express GFP will glow to show that you've had successful delivery," said Katie. Using the fluorescent microscope, she can tell the extent of how successful the process is.

Getting focused: When viewing the bacterially treated cells through the microscope, Katie ran into a design flaw with the plastic dishes in which she incubating her cells. "The plastic was too thick, so we couldn't get clear pictures with the microscopy technique." While Katie put her microscope studies on hold until she could get better plates, she ran other experiments to see the effectiveness of the LLO protein in breaking up, or lysing, the vesicle to release genetic information into the cell. "At higher concentrations of the bacteria with the LLO gene, there's more lysis, which makes sense," said Katie.

Bonus: Katie's lab mate, Saba, has been running simultaneous experiments using luminescence, instead of fluorescence, to measure delivery of genetic material from the bacteria into the immune cells. "Katie is doing the visual part, which is the complement to my work with luminescence," said Saba. Instead of GFP, Saba's bacterial strains contain the gene to produce luciferase, a protein that glows when produced by the immune cells. Using a plate reader, Saba can measure the strength glow which is related to the amount of the protein expression by the mammalian cells.

Future: The finesse comes in modifying the bacterial strains to trigger an appropriate and effective immunotherapeutic response that engineers can control. "We'll see if the changes we're making to the plasmids are resulting in any difference in the efficient delivery of the cargo from the bacterial vectors to the mammalian cells," said Saba. "We have to find out how to control the immune response so that the body fights the cancer cells specifically," Katie said.

Name: Nicholas Horelik, ChBE'09
Project title: Biofabrication of Functional Nanostructures with Genetically Modified Viral Nanotemplates
Advisor: Assistant Professor Hyunmin Yi

What it's all about: The tobacco mosaic virus (TMV) is a plague to farmers of tobacco, tomato, pepper and cucumbers. In the wild, the RNA-based virus discolors leaves, creating a mosaic of light and dark green patches, and subsequently destroys crops. But in the lab, the small, rod-shaped viruses serve as "nanotemplates"—or the frameworks upon which engineers can bind different molecules and other functional nano-sized chemical groups. TMVs can be genetically and chemically modified to be used for purposes such as monitoring of different biological threats. "These viruses are actually biologically derived nanotubes with great potential," said Assistant Professor Hyunmin Yi. "Each particle may have its own information as a biosensor." With more than 2,000 binding sites per TMV, engineers could create a vast library of biosensors, all housed on the same chip. As TMVs are organic structures which occur in nature, engineers can more precisely control the assembly and production than in the development of other silica- or carbon-based structures—opening a new, and potentially superior, avenue of nanotechnology research.

How it works: To prepare his samples, first Nick must prepare the gold-layered silicon chips to which the TMVs will attach. After a thorough chemical rinsing to rid the chips of organic materials and several minutes in a plasma etcher to destroy chemical residues, the chips are allowed to incubate in the TMV solution overnight. Because of the genetic modifications to the virus they bind to the gold surface, under the right conditions producing a single viral layer on the chip. "It's really simple, with one step, which is really attractive. Previous studies have lined up viruses in one layer with multiple coating on coating steps," said Nick, who verifies the specificity and precision of the binding with detailed Atomic Force Microscopy (AFM) images. With the successful achievement of this self-assembly procedure, those same genetic modifications that allow the TMVs to bind to the gold surface can then be utilized to create a chip with various bound metals, which would lead to highly controlled design and fabrication of active nanodevices such as nanoswitches. Currently, Nick is fine-tuning the binding procedure to get more fundamental understanding of the binding behavior, as well as investigating metallization.

Bonus: "I'm getting quite good at the AFM. It's a really common tool that everyone uses in the field, so it's good to know how to use it," said Nick.

Future: Given the flexibility of the TMV technology, there are many different research projects to pursue. Nick and Professor Yi's group are developing high capacity biosensors and nanoswitches based on these potent nanotemplates, which may make major impact on a number of fields.

Lessons learned: "Professor Yi really let me do the experiments I wanted to try," said Nick. "This is my first time in a lab setting and I've learned so much."