UT Chem/Biochem Dept

The 2011-2012 Beckman Scholars: Samuel David Bienvenu

Faculty Mentor: Professor Jason B. Shear Length of term: Summer 11, Fall 11, Spring 12, Summer 12 Honors & Awards:University Honors (Fall 09, Spring 10, Fall 10, Spring 12); Robert Dedman Merit Scholarship (2009); Walter E. Millett Endowed Undergraduate Scholarship in Physics (2011). Publications: in progress.. Where is he now? Graduated with Bachelor of Science (Physics), May 2013. Sam is working as a Lab Research Assistant in the Dept of Neuroscience at UT Austin. How can I contact him? sam.bienvenu at gmail.com

Beckman research project in the Shear group

. Multiphoton fabrication as a technique to confine bacteria into biofilm-like densities

Cystic fibrosis (CF), one of the most common life-shortening genetic disorders in the world, affects over 30,000 individuals in the U.S. alone. Sufferers of this disease have half the average life expectancy due to the combination of their weakened immune system and an adaptation of the ubiquitous bacterium, Pseudomonas aeruginosa. Despite continuous antibiotic treatment, bacterial communities, called biofilms, form within host lungs, acquiring social adaptations that enhance bacterial virulence. To study mechanisms of biofilm adaptations, new strategies are required for systematically evaluating the transition of bacterial behavior in small clusters of cells. The Shear laboratory has pioneered the use of multiphoton lithography to create picoliter-sized chambers from photocrosslinked protein to grow small populations of cells from individual bacteria. These chambers are characterized by biologically relevant conditions, including rapid transport of nutrients, signals, and waste products, providing a means to assess the role of various parameters (e.g., chamber size and geometry) in the onset of bacterial group behaviors, including antibiotic resistance and quorum sensing. The protein-based materials that comprise chamber walls also have been shown to have environmentally sensitive properties, including the ability to under size and shape changes with modifications to solution content . My current work is directed at extending the environmental sensitivity of these materials by developing strategies to alter the volume of photocrosslinked casein, a protein found in cow's milk, by later adding rennin, the enzyme used to curdle milk in cheese production. This technique could provide a new, biologically safe, responsive feature to micro-fabricated structures and may lead to capabilities for rapidly modulating the volumes within which bacteria are contained to more effectively probe density-dependent behaviors of P. aeruginosa.

During my first summer as a Beckman Scholar, I learned to fabricate 3D protein (bovine serum albumin) structures with resolution of less than one micron. A laser focused through a microscope objective excites photosensitizers in a protein solution. Sub-micron resolution is achieved by passing the laser through a very large numerical aperture and narrowing the beam down to a very tight focus. Fabrication is dependent on the intensity of the beam since two photons of the wavelength used are necessary to excite the photosensitizer. The high intensity required restricts reactions to the focal volume. Localized excited-state processes create solids that are written into structures by moving the microscope stage.

Such structures are useful experiments with the bacterium Pseudomonas aeruginosa. These microscopic chambers can be used to trap and confine individual bacterium by leaving a small opening that is later swollen shut by increasing the temperature of the system. The structures are held together by cross-linking proteins and are thus porous enough to allow bacterial growth to proceed normally such that a single trapped bacterium can become a spatially isolated and dense aggregation of bacterial cells. By allowing high densities of bacteria to grow in these chambers, spatially arranged and confined clusters of biofilm like densities are created. Previous experiments have shown that these high-density bacterial populations show antibiotic resistance (as normally exhibited by a P. aeruginosa biofilm), and plans are in place to measure the dependence of that resistance on the rate of flow of media through the chamber. I also learned to set up and operate sterile, contained flow systems for my experiments. I have also learned to create masks for the laser fabrication system in order to design any shape structure I desire. Experiments were designed to collect data concerning the dependence of antibiotic resistance on flow rate, and plans were made to design a chamber in which a low density bacterial population is adjacent to a high density population and measure changes in the low density's death rate in order to discover properties of the signaling molecule which mediates the transcriptional change required for P. aeruginosa to exhibit antibiotic resistance.

During the school year, I explored the innate environmental responsiveness of our lab's microfabricated structures. So far, various structures fabricated in the Shear Lab are responsive to changes in the temperature, pH, and ionic strength of their environment. Some structures have also been shown to respond to illumination with a fluorescent lamp as well as re-exposure to the fabrication beam. In addition, I spent many months optimizing and testing a protein solution, which I formulated in order to fabricate structures composed of casein isoforms.

My summer work aimed to add enzymatic responsiveness to the Shear Lab's toolkit of environmental responsiveness. Structures are fabricated by photocrosslinking casein, a protein found in cow's milk, in order to induce dynamic response by later adding rennin, the enzyme used to curdle milk in cheese production. The mixed casein isoforms used to make these structures form micelles: the hydrophobic isoforms of casein cluster together, and the amphiphilic isoform, _-casein, interfaces between water and the hydrophobic clusters.

Under normal circumstances, the casein micelles are coated with the charged groups of _-casein and electrostatically repel one another. Rennin acts by cleaving the charged groups off of _-casein. The removal of the charged groups abolishes the repulsion between micelles and leads the micelles to hydrophobically associate. Experimentation successfully shows that adding rennin collapses a structure fabricated from casein by inducing hydrophobic interactions between the incorporated micelles. In summary, the casein structures collapsed in response to pHs near the isoelectric point of _-casein and in response to the addition of rennin (2 mg/mL). Casein structures swelled in response to basic pHs and in response to illumination from a fluorescent lamp. No structural changes were found in response to temperatures up to 60¡C or re-scanning of fabrication laser.