20.109(S08):Module 1

Module 1

Instructors: Bevin Engelward and Agi Stachowiak

TA: David Weingeist

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.

We gratefully acknowledge 20.109 instructor Natalie Kuldell for her crucial work developing and teaching previous iterations of this module.

Recombocell image from Dominika Wiktor of the Engelward Lab

A schematic overview of the module.

Timetable of the module.

Lab handouts

Day 1: DNA engineering using PCR (you will also need weblinks, below)

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

Module 1 lab report schedule and guidelines

DNA engineering web links

Engelward lab resources: https://web.mit.edu/bevin/www/UltiMouse/

pCX-EGFP plasmid map: https://web.mit.edu/bevin/www/UltiMouse/pCX-EGFP.pdf File:Macintosh HD-Users-nkuldell-Desktop-pCX-EGFP.doc

ORF finder: http://www.ncbi.nlm.nih.gov/gorf/gorf.html

NCBI: http://www.ncbi.nlm.nih.gov/

Cybergene: http://www.cybergene.se/primer.html

New England Biolabs: http://www.neb.com/nebecomm/default.asp

References

Note: PDF reprints are provided below within the context of fair use. Please obtain copies from the publisher if appropriate.

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 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
   PDF reprint

Notes for Teaching Faculty

TA notes, mod 1