Difference between revisions of "20.109(F16):Module 1"

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==Overview==
 
==Overview==
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In this experimental module, with a view to quantify DNA repair by homologous recombination, you will modify the gene for EGFP (Enhanced Green Fluorescent Protein) to truncate the protein it encodes. Cells expressing the full-length protein glow green when exposed to light of the appropriate wavelength. You will be designing and then creating an expression vector to delete the first 32 amino acids of EGFP. Cells transfected with your expression vector should not glow green, a prediction you will test. You will also test whether this N-terminally truncated EGFP can recombine with a C-terminally truncated version to regenerate full length EGFP ''in vivo''. Finally, you will have the opportunity to suggest changes to the experimental protocol that will increase the frequency of green cells in which there has been an inter-plasmid recombination event.  We will then choose a few variables to test on the final day of the experiment.
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In this module you will measure DNA repair using two assays: the comet chip and immuno-fluorescence.  Your first task is to critically think through the development of the comet chip assay and determine which conditions provide the best results. To this end, you will consider variables that effect cell loading into the microwells of the comet chip and variables that effect the readout of the results. The data you collect will be used to propose a high-throughput comet chip assay for commercial use.
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Next, you will use the comet chip assay to assess the effect of chemicals and ultraviolet-irradiation on DNA repair.  Specifically, you will assess the base excision repair pathway and the nucleotide excision repair pathway. Last, you will examine the effect of gamma-irradiation on double strand breaks using an immuno-fluorescence approach.
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[[Image:Fa16 M1 overview.png|thumb|left|250px|Experimental overview for Module 1]]
  
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[[Image:Be109recombomouse.jpg|thumb|left|250px|Recombocell image from Dominika Wiktor of the Engelward Lab]]
 
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[[Image:Experimental Overview.jpg|thumb|right|450px|A schematic overview of the Module 1.]]
 
 
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Revision as of 20:34, 28 June 2016

20.109(F16): Laboratory Fundamentals of Biological Engineering

Engelward PNAS 2006.png

Schedule Fall 2016        Announcements        Assignments        Homework        Communication
       1. Measuring Genomic Instability        2. Manipulating Metabolism        3. Engineering Biomaterials              

Module 1

Lecturer: Bevin Engelward
Instructors: Noreen Lyell, Leslie McClain and Maxine Jonas

TA:
Lab manager: Hsinhwa Lee

Overview

In this module you will measure DNA repair using two assays: the comet chip and immuno-fluorescence. Your first task is to critically think through the development of the comet chip assay and determine which conditions provide the best results. To this end, you will consider variables that effect cell loading into the microwells of the comet chip and variables that effect the readout of the results. The data you collect will be used to propose a high-throughput comet chip assay for commercial use.

Next, you will use the comet chip assay to assess the effect of chemicals and ultraviolet-irradiation on DNA repair. Specifically, you will assess the base excision repair pathway and the nucleotide excision repair pathway. Last, you will examine the effect of gamma-irradiation on double strand breaks using an immuno-fluorescence approach.


Experimental overview for Module 1


Lab links: day by day

M1D1: DNA engineering using PCR
M1D2: Clean and cut DNA
M1D3: Agarose gel electrophoresis
M1D4: Ligation & transformation
M1D5: Examine candidate clones and tissue culture
M1D6: Lipofection
M1D7: Data analysis

Assignments

DNA engineering summary
DNA engineering mini-presentation

Useful online resources

References

  1. DNA double-strand break repair: From mechanistic understanding to cancer treatment
    DNA Repair 2007
    Thomas Helleday, Justin Lo, Dik C. van Gent, Bevin P. Engelward
    URL
    Sample Animation Animations were made by Justin Lo (BE class of '08), a former UROP student in Professor Engelward's laboratory!
  2. Homologous recombination as a mechanism of carcinogenesis
    Biochim Biophys Acta 21 March 2001
    Bishop AJ and Schiestl RH
    URL
  3. Rad51-deficient vertebrate cells accumulate chromosomal breaks prior to cell death
    EMBO J 15 January 1998
    E Sonoda, M S Sasaki, J M Buerstedde, O Bezzubova, A Shinohara, H Ogawa, M Takata, Y Yamaguchi-Iwai, and S Takeda M
    URL
  4. NEBuffer Performance Chart with Restriction Enzymes
    Old buffer system: URL
    New buffer system: URL

Notes for teaching faculty

TA notes, M1

F16 notes for orientation day
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