20.109(F08):Module 2: Difference between revisions

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'''Instructors:''' [[Natalie Kuldell]], [[User:AgiStachowiak| Agi Stachowiak]]
'''Instructors:''' [[Natalie Kuldell]], [[User:AgiStachowiak| Agi Stachowiak]]


'''TA:''' [[]]
'''TA:''' [http://openwetware.org/wiki/User:Brian_Belmont 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 strains using a microarray. Our individual experiments may identify targets for 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.  
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, <i>S. cerevisiae,</i> 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.  


[[Image:Macintosh HD-Users-nkuldell-Desktop-ExpressionEng coverart S07.jpg|thumb|500 px|center| SAGA image from F.Winston<br> Mode of action model from P. Schultz <br> Mol Cell. 2004 Jul 23;15(2):199[[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15260971&query_hl=4&itool=pubmed_docsum]]<br> Microarray image from N. Kuldell<br>]]
[[Image:Macintosh HD-Users-nkuldell-Desktop-ExpressionEng coverart S07.jpg|thumb|500 px|center| SAGA image from F.Winston<br> Mode of action model from P. Schultz <br> Mol Cell. 2004 Jul 23;15(2):199[[http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15260971&query_hl=4&itool=pubmed_docsum]]<br> Microarray image from N. Kuldell<br>]]


[[20.109(F08): Start-up expression engineering| Module 2 Day 1: Start-up expression engineering]]<br>
[[20.109(F08): Mod 2 Day 1 Protein engineering with PCR| Day 1: Protein engineering with PCR]]<br>
[[20.109(F08): Yeast transformation| Module 2 Day 2: Yeast transformation]]<br>
[[20.109(F08): Mod 2 Day 2 Yeast transformation| Day 2: Yeast transformation]]<br>
[[20.109(F08): Colony PCR| Module 2 Day 3: Colony PCR]] <br>
[[20.109(F08): Mod 2 Day 3 Colony PCR and journal article discussion| Day 3: Colony PCR, Journal article discussion]] <br>
[[20.109(F08): Western Analysis| Module 2 Day 4: Western Analysis ]]<br>
[[20.109(F08): Mod 2 Day 4 SDS-PAGE, screen for phenotypes| Day 4: SDS-PAGE, screen for phenotypes]]<br>
[[20.109(F08): Probe Western| Module 2 Day 5: Probe Western ]]<br>
[[20.109(F08): Mod 2 Day 5 Probe western, isolate RNA| Day 5: Probe Western, isolate RNA]]<br>
[[20.109(F08): Screen for phenotypes, isolate RNA| Module 2 Day 6: Screen for phenotypes, isolate RNA]] <br>
[[20.109(F08): Mod 2 Day 6 Journal Club I| Day 6: Journal Club I]] <br>
[[20.109(F08): cDNA synthesis and microarray| Module 2 Day 7: cDNA synthesis and microarray]] <br>
[[20.109(F08): Mod 2 Day 7 cDNA synthesis and microarray| Day 7: cDNA synthesis and microarray]] <br>
[[20.109(F08): Microarray data analysis| Module 2 Day 8: Microarray data analysis]]<br>
[[20.109(F08): Mod 2 Day 8 Microarray data analysis| Day 8: Microarray data analysis]]<br>
direct link to writeup <br>
direct link to oral presentation


[[20.109(F08): Protein engineering research article| Protein Engineering Research Article Guidelines]]<br>
[[20.109(F08):Guidelines for oral presentations| Guidelines for Journal Club Presentations]]
==Notes for Teaching Faculty==
[[20.109(F08): TA's notes for module 2| TA notes, mod 2]]
[[20.109(F08): TA's notes for module 2| TA notes, mod 2]]

Latest revision as of 06:05, 16 August 2008


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

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