20.109(F08):Module 2

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20.109(F08): Laboratory Fundamentals of Biological Engineering

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Module 2

Instructors: Natalie Kuldell, Agi Stachowiak

TA: Brian Belmont

Faced with the considerable challenge of packing more DNA into a cell, nature added proteins that reversibly compact the helix. The DNA can wind around these histone proteins, forming nucleosomes, that can then wind around each other to form chromatin. Other protein complexes modify and remodel the chromatin, making the DNA accessible for reading and copying. “SAGA” is a chromatin remodeling complexes in the model experimental yeast, S. cerevisiae, but it turns out not every protein in SAGA is needed for the yeast to survive. In this experiment we will modify one of the SAGA-subunits in the yeast genome and then ask how the resulting yeast, though alive, is affected. We will look for phenotypes that might indicate crippled functions and we will compare gene expression in the parent strain to each tagged strain using a microarray. Our individual experiments may identify genes controlled by particular SAGA subunits while our class data may reveal genes that are commonly regulated by this remodeling complex. Given the structural information for SAGA that is recently available, we can hope to map our findings onto the complex and better understand the delicate balance between chromatin remodeling and gene expression.

SAGA image from F.Winston
Mode of action model from P. Schultz
Mol Cell. 2004 Jul 23;15(2):199[[1]]
Microarray image from N. Kuldell

Day 1: Protein engineering with PCR
Day 2: Yeast transformation
Day 3: Colony PCR, Journal article discussion
Day 4: SDS-PAGE, screen for phenotypes
Day 5: Probe Western, isolate RNA
Day 6: Journal Club I
Day 7: cDNA synthesis and microarray
Day 8: Microarray data analysis

Protein Engineering Research Article Guidelines
Guidelines for Journal Club Presentations

Notes for Teaching Faculty

TA notes, mod 2