20.109(F14):Module 1

Module 1

Instructors: Bevin Engelward, Shannon Hughes, Agi Stachowiak

TA:

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

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
Ligation & Transformation and CometChip prep
Examine candidate clones and CometChip Tissue Culture
Lipofection and CometChip Analysis
Day 7: FACS analysis

Assignments

Abstract and data summary: Assignment description

Plasmid construction methods section: Assignment description

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, mod 1 S12 notes for orientation day