BE.109:DNA engineering

Module 1

Instructors: Bevin Engelward and Natalie Kuldell

TA: Yoon Sung Nam

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.

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. Pathways for mitotic homologous recombination in mammalian cells
   Mutation Research 27 November 2003
   Thomas Helleday
   URL
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