Suzanne Fleiszig, Professor

Closed (1) Genetic knockouts of Pseudomonas aeruginosa

Closed. This professor is continuing with Spring 2017 apprentices on this project; no new apprentices needed for Fall 2017.

Pseudomonas aeruginosa is the most common cause of contact lens-mediated microbial keratitis even in healthy patients. Our lab has found that P. aeruginosa can survive and replicate inside of epithelial cells during corneal infection and we have identified multiple virulence genes that are involved in this process. To adequately test these genes, various genetic knockouts need to be generated, such as deleting the whole gene, or mutating an active site amino acid. Mutants will be tested by a number of different assays such as growth curves, real time PCR, human cell culture infection, and animal models of disease.

Students will learn basic molecular cloning techniques including PCR, restriction digests, use of laboratory strains of E. coli, and allelic exchange in Pseudomonas aeruginosa. Depending on the mutants students select to make (which can be tailored to their interests), function will be tested by growth assays, Western blots, infections of cultured human cells, microscopy, or students may assist in animal model experiments conducted by the supervising postdoc. Students are expected to present their progress in lab meeting. Completion of a mutant with characterized phenotype has typically led to authorship on our lab's publications.

Day-to-day supervisor for this project: Abby Kroken, Post-Doc

Qualifications: Basic knowledge of microbiology is required, as well proficiency with scientific notation, calculating molarity and concentrations, diluting concentrated solutions, and basic use of Microsoft Excel--completion or concurrent enrollment in general chemistry and biology classes should suffice. Previous lab experience such as the use of pipettes and sterile technique is good, but not required. Ideally we would like to recruit students who are sophomores or juniors who are interested in a multi-year commitment and are able to apply for summer fellowships in 2018 (examples include the American Society for Microbiology undergraduate research fellowship or SURF: surf.berkeley.edu.) This project benefits from small blocks of time each day (2-3 hours/day) instead of one large block of time, and it will be necessary to schedule the hours between 9a-5p for proper attention to training.

Weekly Hours: 9-12 hrs

Related website: http://fleiszig.vision.berkeley.edu

Open (2) Fluorescent reporter strains of Pseudomonas aeruginosa for monitoring gene expression during corneal infection

Open. Apprentices needed for the fall semester. Please do NOT contact faculty before September 11th (the start of the 4th week of classes)! Enter your application on the web beginning August 16th. The deadline to apply is Tuesday, August 29th at 8 AM.

Pseudomonas aeruginosa is the most common cause of contact lens-mediated microbial keratitis, even in healthy patients. Our lab has developed a mouse model of infection using specially designed contact lenses, in which we observe the development of P. aeruginosa-derived keratitis over time. Using confocal microscopy, we are able to image P. aeruginosa on the contact lens and interacting with the corneal epithelium prior to and during an actual infection. One major advantage of this technique is that we can investigate bacterial gene expression, using a series of P. aeruginosa reporter strains, in real-time to better understand the changes necessary for P. aeruginosa to transition from living on the contact lens to invading the corneal epithelium. To gain a comprehensive picture of what conditions bacteria must adapt to in order to survive on a contact lens in a mouse eye, we are creating a large set of reporter strains in P. aeruginosa, that respond to different stressors by inducing fluorescent protein production. Reporter strains must be tested in vitro by a variety of assays to ensure that they respond appropriately to the conditions we intend them to respond to, and to develop strategies to quantify these responses.

Student will learn basic molecular cloning techniques including PCR, restriction digests, and use of laboratory strains of E. coli to create new reporter strains and optimize the current fluorescent protein reporter system. Student will then assist with testing these reporters in vitro by exposing bacteria to a variety of physiological stressors and monitoring fluorescence on a plate reader as well as by microscopy. Student may also have the opportunity to create genetic knockouts of P. aeruginosa to further investigate the roles of key genes in mediating an appropriate transcriptional response to the ocular surface. Student may assist in animal model experiments conducted by the supervising postdoc and is expected to present his or her progress in lab meeting.

Day-to-day supervisor for this project: Melinda Grosser, Post-Doc

Qualifications: Basic knowledge of microbiology is required, as well proficiency with scientific notation, calculating molarity and concentrations, diluting concentrated solutions, and basic use of Microsoft Excel--completion or concurrent enrollment in general chemistry and biology classes should suffice. Previous lab experience such as the use of pipettes and sterile technique is useful, but not required. Ideally we would like to recruit students who are sophomores or juniors who are interested in a multi-year commitment and able to apply for summer fellowships in 2018 (examples include the American Society for Microbiology undergraduate research fellowship or SURF: surf.berkeley.edu.) This project benefits from small blocks of time each day (2-3 hours/day) instead of one large block of time, and it will be necessary to schedule the hours between 9a-5p for proper attention to training. Please consider our scheduling needs when applying.

Weekly Hours: 9-12 hrs

Related website: http://vision.berkeley.edu/fleisziglab/

Closed (3) Adsorption of protective glycoprotein (FRIT) from Human tears to contact lens

Closed. This professor is continuing with Spring 2017 apprentices on this project; no new apprentices needed for Fall 2017.

Pseudomonas aeruginosa is the most clinically important pathogen in human microbial keratitis (infection of the cornea), especially for contact lens associated keratitis. Using a mouse model of infection, we previously reported that twitching motility, which is a surface-associated bacterial movement mediated by Type IVa pili, contributes to corneal disease. Our lab recently discovered that a glycoprotein FRIT in human tears suppresses twitching motility, and also protects murine eyes from P. aeruginosa infection in vivo. Interestingly, artificial tears, which have all the major proteins of human tears, do not inhibit twitching motility. Therefore, the goal of this project is to explore adsorption of FRIT or FRIT with artificial tears to contact lens materials and how that impacts P. aeruginosa twitching at the material surface. This could lead to further understanding about defensive action of FRIT and provide novel strategies to combat contact lens associated eye infection.

Students will begin with biochemical-based experiments, purify proteins from human subject samples, analyze proteins by SDS-PAGE and ELISA, and then assess the protein’s activity on inhibiting bacterial twitching motility. They will also learn fluorescence methods to observe FRIT adsorption on contact lens using microscopy, and quantify fluorescence signal on contact lens.

Day-to-day supervisor for this project: Jianfang Li, Post-Doc

Qualifications: Interest in learning protein purification and analysis and microscopy are needed. Students having good computer skills would be perfect. Previous lab experience is strongly desired, such as experience using micropipettes, sterile technique, scientific notation, and calculating molarity and concentrations.

Weekly Hours: more than 12 hrs

Related website: http://fleiszig.vision.berkeley.edu

Closed (4) Studying Pseudomonas aeruginosa infection using corneal epithelial cells

Closed. This professor is continuing with Spring 2017 apprentices on this project; no new apprentices needed for Fall 2017.

Keratitis is a devastating disease affecting humans that frequently wear contact lenses. Pseudomonas aeruginosa is found to be the main causative agent of infection of human corneal epithelial cells in such cases. To analyze the steps in which P. aeruginosa infection spreads, we look to determine P. aeruginosa's mechanism of host cell exit. P. aeruginosa can act as an invasive pathogen, where it can replicate within its host and finally exit to continue its infection cycle. This project will be using human corneal epithelial cell cultures and infection assays with multiple P. aeruginosa strains and mutants. We aim to answer the main question: how does P. aeruginosa exit host cells after initial infection? In order to answer this, we will be utilizing various laboratory techniques to determine both functional components in P. aeruginosa that are important for exit, as well as any observe and quantify any variability of host cells (such as changes in cytoskeleton, membrane blebbing, cell death).

Students will learn how to culture human-derived epithelial cells as well as maintain them. Basic microbiology techniques and assays (including infection assays) will be utilized. If a student is successful, they will continue with confocal and wide-field microscopy experiments, and possibly extend their work into mouse models.

Day-to-day supervisor for this project: Vincent Nieto, Post-Doc

Qualifications: An interest in learning microbiology and cell culture techniques are a must. Previous lab experience is desired (academic training labs are fine). Be experienced in using math related to chemical composition and for media production (growth media, buffers, et cetera). Have at least 1 large block of time per week (at least 5 hours).

Weekly Hours: more than 12 hrs