20.109(F07):Module 2: Difference between revisions

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(New page: {{Template:20.109(F07)}} <div style="padding: 10px; width: 640px; border: 5px solid #666699;"> ==Module 1== '''Instructors:''' Drew Endy and Natalie Kuldell '''TA:''' In this...)
 
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==Module 1==
==Module 2==


'''Instructors:''' [[Drew Endy]] and [[Natalie Kuldell]]
'''Instructors:''' [[Natalie Kuldell]] and [[User:AgiStachowiak| Agi Stachowiak]]


'''TA:'''  
'''TA:'''[[User:AliceLo| Alice Lo]]


In this experiment, we will consider the genome of a virus, namely the bacteriophage M13. M13 is a self-assembling nano-machine with a compact genome that has been optimized by evolution to commandeer its bacterial host. Approximately 1000 new viruses are generated from a single infection event. Imagine harnessing this production. What could we build and what  natural processes could we better understand? One approach we’ll take is to modify the existing genome in a subtle but useful way, namely by adding a peptide-tag that can be presented on the bacteriophage coat. We’ll examine how this modification affects the coat protein’s expression and overall phage production. Another approach we’ll take is to start from scratch, undertaking a full throttle redesign of the bacteriophage genome. We’ll employ a commercial DNA synthesis company to compile the redesigned genomic program and then we’ll see if it encoded infective M13 and if the genome of the bacterial host affects bacteriophage production. Through these investigations we’ll ask: is nature’s M13 genome “perfect” or can we do better?  
In this experiment, we will consider unintended and unpredicted effects of an experimental perturbation. Our goal is a precise one, namely to silence gene expression of a measurable gene, luciferase, using RNA interference (RNAi). Each group will begin by designing a short interfering RNA (siRNA) against luciferase, but as we'll see, siRNAs can vary in efficacy and specificity. After transfecting a mammalian cell line with the siRNA you’ve designed and a reporter plasmid, we will evaluate the silencing using a luciferase assay and microarray technology. The first assay evaluates the efficacy of the siRNA in silencing. The second assay gives genome-wide expression data to reveal the specificity of your siRNA for the gene you’ve targeted. Through this combined approach, we'll assess the balance of targeted and off-target effects.
   
[[Image:Macintosh_HD-Users-nkuldell-Desktop-sprinkler.jpg|400px|center]]


[[Image:Macintosh HD-Users-nkuldell-Desktop-GnmEng coverart S07.jpg|thumb|500px|center|M13-coated coli from M. Russel<br> Map of M13 genome from M. Blaber]]
[[20.109(F07): siRNA design | Module 2 Day 1: siRNA design and introduction to cell culture]]<br>
[[20.109(F07): Transfection | Module 2 Day 2: Transfection]]<br>
[[20.109(F07): Luciferase assays and RNA prep| Module 2 Day 3: Luciferase assays and RNA prep]]<br>
[[20.109(F07): Journal article discussion| Module 2 Day 4: Journal article discussion]]<br>
[[20.109(F07): cDNA synthesis and microarray| Module 2 Day 5: cDNA synthesis and microarray]]<br>
[[20.109(F07): Microarray data analysis| Module 2 Day 6: Microarray data analysis]]<br>
[[20.109(F07): Mod 2 Day 7| Module 2 Day 7: Reports due]]<br>
[[20.109(F07): Module 2 oral presentations| Module 2 Day 8: Oral Presentations]]<br>
direct link to [[20.109(F07): Expression engineering report| writing assignment]]<br>


[[20.109(S07):Start-up genome engineering | Module 1 Day 1: Start-up genome engineering]]<br>
[[20.109(F07): TA's notes for module 2| TA notes, mod 2]]
[[20.109(S07): Agarose gel electrophoresis| Module 1 Day 2: Agarose gel electrophoresis]]<br>
[[20.109(S07): DNA ligation and bacterial transformation| Module 1 Day 3: DNA ligation and bacterial transformation]]<br>
[[20.109(S07): Examine candidate clones| Module 1 Day 4: Examine candidate clones]]<br>
[[20.109(S07): Western analysis| Module 1 Day 5: Western analysis]]<br>
[[20.109(S07): Probe western| Module 1 Day 6: Probe western]]<br>
direct link to [[20.109%28S07%29:_Genome_engineering_essay]]<br>
direct link to [[20.109:Module 1:RefactorM13| M13 refactoring workpage]]<br>
direct link to [http://parts.mit.edu/r/parts/partsdb/part_info.cgi?part_name=BBa_M1307| hard info for BBa_M1307]
 
[[20.109(S07): TA's notes for module 1| TA notes, mod 1]]

Latest revision as of 07:11, 2 September 2007


20.109(F07): Laboratory Fundamentals of Biological Engineering

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Module 2

Instructors: Natalie Kuldell and Agi Stachowiak

TA: Alice Lo

In this experiment, we will consider unintended and unpredicted effects of an experimental perturbation. Our goal is a precise one, namely to silence gene expression of a measurable gene, luciferase, using RNA interference (RNAi). Each group will begin by designing a short interfering RNA (siRNA) against luciferase, but as we'll see, siRNAs can vary in efficacy and specificity. After transfecting a mammalian cell line with the siRNA you’ve designed and a reporter plasmid, we will evaluate the silencing using a luciferase assay and microarray technology. The first assay evaluates the efficacy of the siRNA in silencing. The second assay gives genome-wide expression data to reveal the specificity of your siRNA for the gene you’ve targeted. Through this combined approach, we'll assess the balance of targeted and off-target effects.

Module 2 Day 1: siRNA design and introduction to cell culture
Module 2 Day 2: Transfection
Module 2 Day 3: Luciferase assays and RNA prep
Module 2 Day 4: Journal article discussion
Module 2 Day 5: cDNA synthesis and microarray
Module 2 Day 6: Microarray data analysis
Module 2 Day 7: Reports due
Module 2 Day 8: Oral Presentations
direct link to writing assignment

TA notes, mod 2

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