Restriction Digestion Lab Report
BIO 219
Group 1
Section 66
October 19, 2012
The extraction of purified DNA from A. fischeri by restriction digestion using
Sal I enzyme and pGEM for shotgun cloning
Introduction:
The ultimate goal of this experiment is to isolate the lux operon, a targeted piece of DNA that causes bioluminescence, from Aliivibrio fischeri and insert it into the DNA of Escherichia coli in order to make it glow. A. fischeri is a gram-negative bacteria which participates in a symbiotic relationship with many marine organisms (Perry et al., 2005). This particular bacterium has the feature of bioluminescence, which is regulated by a ~nine-kilobase fragment of their chromosomal DNA, the lux operon (Slock et al., 2000). The lux operon contains a group …show more content…
A 0.8% agarose gel was used as the medium for the DNA solutions because it has a better separation of large DNA fragments due to the larger pores throughout the gel. The electrodes on each end of the apparatus allowed the fragments to migrate across the gel. The opposite end of the DNA-containing wells has a positively charged electrode that attracts the DNA, which is negatively charged due to its phosphate backbone. Electrophoresis confirmed whether or not the DNA samples were digested properly by observing the lengths of the fragments in accordance to their position on the gel after completion of the …show more content…
2008. Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation. J. Ind. Microbiol. Biotechnol. (35): 1447–1454.
Carnes, E. C., Lopez, D. M., Donegan, N. P., Cheung, A., Gresham, H., Timmins, G. S., &
Brinker, C. J. (2010). Confinement-induced quorum sensing of individual staphylococcus aureus bacteria. Nature Chemical Biology, 6(1), 41-5. doi: http://dx.doi.org/10.1038/nchembio.264
Greenberg, EP, Schaefer, AL, Hanzelka, BL, and Eberhard, A. 1996. Quorum Sensing in Vibrio fischeri: Probing Autoinducer-LuxR Interactions with Autoinducer Analogs. Journal of Bacteriology (178): 2897-2901.
Lyell, N. L., Dunn, A. K., Bose, J. L., & Stabb, E. V. (2010). Bright mutants of vibrio fischeri
ES114 reveal conditions and regulators that control bioluminescence and expression of the lux operon. Journal of Bacteriology, 192(19), 5103-5114. doi: http://dx.doi.org/10.1128/JB.00524-10
Perry, LL, Bright, NG, Carroll, RJ, and Scott, MC. 2005. Molecular characterization
Group 1
Section 66
October 19, 2012
The extraction of purified DNA from A. fischeri by restriction digestion using
Sal I enzyme and pGEM for shotgun cloning
Introduction:
The ultimate goal of this experiment is to isolate the lux operon, a targeted piece of DNA that causes bioluminescence, from Aliivibrio fischeri and insert it into the DNA of Escherichia coli in order to make it glow. A. fischeri is a gram-negative bacteria which participates in a symbiotic relationship with many marine organisms (Perry et al., 2005). This particular bacterium has the feature of bioluminescence, which is regulated by a ~nine-kilobase fragment of their chromosomal DNA, the lux operon (Slock et al., 2000). The lux operon contains a group …show more content…
A 0.8% agarose gel was used as the medium for the DNA solutions because it has a better separation of large DNA fragments due to the larger pores throughout the gel. The electrodes on each end of the apparatus allowed the fragments to migrate across the gel. The opposite end of the DNA-containing wells has a positively charged electrode that attracts the DNA, which is negatively charged due to its phosphate backbone. Electrophoresis confirmed whether or not the DNA samples were digested properly by observing the lengths of the fragments in accordance to their position on the gel after completion of the …show more content…
2008. Photodynamic inactivation of recombinant bioluminescent Escherichia coli by cationic porphyrins under artificial and solar irradiation. J. Ind. Microbiol. Biotechnol. (35): 1447–1454.
Carnes, E. C., Lopez, D. M., Donegan, N. P., Cheung, A., Gresham, H., Timmins, G. S., &
Brinker, C. J. (2010). Confinement-induced quorum sensing of individual staphylococcus aureus bacteria. Nature Chemical Biology, 6(1), 41-5. doi: http://dx.doi.org/10.1038/nchembio.264
Greenberg, EP, Schaefer, AL, Hanzelka, BL, and Eberhard, A. 1996. Quorum Sensing in Vibrio fischeri: Probing Autoinducer-LuxR Interactions with Autoinducer Analogs. Journal of Bacteriology (178): 2897-2901.
Lyell, N. L., Dunn, A. K., Bose, J. L., & Stabb, E. V. (2010). Bright mutants of vibrio fischeri
ES114 reveal conditions and regulators that control bioluminescence and expression of the lux operon. Journal of Bacteriology, 192(19), 5103-5114. doi: http://dx.doi.org/10.1128/JB.00524-10
Perry, LL, Bright, NG, Carroll, RJ, and Scott, MC. 2005. Molecular characterization