Growth Of Yeast Bio-films

By Raykia Koroma

Background InformationThere is a species of yeast cell called Saccharomyces cerevisiae that is commonly known as Baker’s Yeast. In the lab I am working with Saccharomyces cerevisiae has been genetically engineered to express the ALS5 gene from another yeast called Candida albicans. Candida albicans is a fungus that causes yeast infections that infect 10 out 1000 people a year. It also kills hundreds of AIDS victims and cancer victims a year. It only attacks people with suppressed immune systems which means it is an opportunistic pathogen. Candida albicans expresses 6,000 genes one of them is ALS5. ALS genes encode an adhesion glycoprotein. There are 8 different ALS genes: ALS1-ALS7 and ALS9.

Purpose

•I want to know if I express this ALS5 protein would I get a different or better type of biofilm.

• If yes what does it mean?

•Is ALS5 important for the growth of the biofilm?

Materials

•Plates

•Flask

•Incubator

•Autoclave

•YPA agar media

•Csm-URA agar media

•Csm-Complete agar media

•W3031B

•ALS5

•ALS1 Strains

•ALS3

•PADH

•Shaker

•Pipette

•Gloves

•Csm-URA liquid

•Csm- complete liquid

•Spectrophotometer

Methodology• I made 500ml of YPA, Csm- URA and Csm- Complete agar media’s. • Split each media into 4 different flasks that contain 125ml in each with

- 2% agar w/galactose - .3% agar w/galactose- 2% agar w/glucose - .3% agar w/glucose

• Autoclaved them for 25 minutes.• Poured them on plates and left them overnight in a 30 degrees C

incubator.• Streak

-ALS5 on Csm-trp-W3031B on YPED-ALS1 on Csm-URA-ALS3 on Csm –URA -PADH on Csm-URA

• Inoculated the strains into flasks and put them in a 30 degree C shaker• Measure their Optical Density(OD)• If they all reach .5 great but if not then I would have to spin them down for about

10 minutes.• Dilute so that it’s 3.3 x10 6 cells/ml2 using the Dimensional Analysis technique.• Then inoculate on plates that are divided into either 3 or 2 sections:

-ALS1, ALS3 & PADH same plate with glucose only-W3031B & ALS5 same plate with glucose or galactose

• Leave them to grow at room temperature to grow for about a week• Analysis will be based on the appearance of the biofilm

Results •No results as of yet.

•I’m expecting for W3031B and PADH to look like

•ALS1 and ALS3 should be a moderate size and be constitutively on .

•ALS5 should grow better on galactose and should look like

AcknowledgementsDr. Peter Lipke

Janice Lee

Marlyn Gonzales

Raymond Fung

Dr. Sat Bhattacharya

Harlem Children Society Staff

Brooklyn College