20.109(F09):Module 1

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'''Instructors:''' [http://web.mit.edu/be/people/engelward.htm Bevin Engelward], [[Natalie Kuldell]], [[User:AgiStachowiak| Agi Stachowiak]]
'''Instructors:''' [http://web.mit.edu/be/people/engelward.htm Bevin Engelward], [[Natalie Kuldell]], [[User:AgiStachowiak| Agi Stachowiak]]
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'''TA:'''  
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'''TA:''' [http://openwetware.org/wiki/User:Michelle_R._Sukup_Jackson Michelle Sukup Jackson]
In this experimental module 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.
In this experimental module 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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[[Image:Be109recombomouse.jpg|thumb|left|200px|'''Recombocell image from Dominika Wiktor of the Engelward Lab''']]
[[Image:Be109recombomouse.jpg|thumb|left|200px|'''Recombocell image from Dominika Wiktor of the Engelward Lab''']]
[[Image:Experimental Overview.jpg|thumb|right|350px|'''A schematic overview of the module.''']]
[[Image:Experimental Overview.jpg|thumb|right|350px|'''A schematic overview of the module.''']]
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==Lablinks: day by day==
==Lablinks: day by day==
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[[20.109(F09): Mod 1 Day 8 FACS analysis| Day 8: FACS analysis]]<br>
[[20.109(F09): Mod 1 Day 8 FACS analysis| Day 8: FACS analysis]]<br>
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[[20.109(F09):DNA engineering lab report guidelines| DNA engineering progress report guidelines]]<br>
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[[20.109(F09):DNA engineering "Progress Report"| DNA engineering progress report guidelines]]<br>
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[[20.109(F09):DNA engineering memo guidelines| "memo" guidelines]]
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[[20.109(F09):DNA engineering "Memo"| "Memo" guidelines]]
==References==
==References==

Current revision

20.109(F09): Laboratory Fundamentals of Biological Engineering

Home        People        Schedule Fall 2009        Assignments        Lab Basics        OWW Basics       
DNA Engineering        System Engineering        Biomaterials Engineering              

Contents

Module 1

Instructors: Bevin Engelward, Natalie Kuldell, Agi Stachowiak

TA: Michelle Sukup Jackson

In this experimental module 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.

Recombocell image from Dominika Wiktor of the Engelward Lab
Recombocell image from Dominika Wiktor of the Engelward Lab
A schematic overview of the module.
A schematic overview of the module.


Lablinks: day by day

Day 1: DNA engineering using PCR
Day 2: Clean and cut DNA
Day 3: Agarose gel electrophoresis
Day 4: DNA ligation and bacterial transformation
Day 5: Examine candidate clones
Day 6: Restriction map and tissue culture
Day 7: Lipofection
Day 8: FACS analysis

DNA engineering progress report guidelines
"Memo" guidelines

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

Notes for Teaching Faculty

TA notes, mod 1
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