MBL1-4 - MBL 1-Cloning the lux Operon from Vibrio fischeri...

Info iconThis preview shows pages 1–3. Sign up to view the full content.

View Full Document Right Arrow Icon
MBL 1 -Cloning the lux Operon from Vibrio fischeri Name: Nick Benzschawel Partner: Tony Desotell Section: 301 3/27/09 Overall Rational
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
The gram-negative bacterium Vibrio fischeri contains a set of genes known as the lux operon that when transcribed has the ability to produce light, a process known as bioluminescence. V. fischeri has been identified as a symbiont of several species of fish and squid. Most noteworthy is the relationship it has with the Hawaiin bobtail squid Euprymna scolopes in which the bacteria colonize a special light organ designed specifically for culturing V. fischeri . It has been proposed that light produced by these bacteria helps to camouflage the squid when it hunts at night by mimicking the moon light and thus thwarting potential predators from below. The bioluminescence produced by V. fischeri has recently been applied as a versatile tool in a wide array of industries. For example, in the biotech industry bioluminescence is used to detect gene transfers and metoblic activity, while the food industry it is used to detect food contaminants and to monitor the presence of antibiotics in cultures used for cheese and yogurt. In the medical community bioluminescence has been substituted for radioactive materials as a means of immonassay detection. Lastly, bioluminescence has proven to be a vital tool in many environmental applications such as toxicity and water pollution monitoring. It is obviously very important to understand how this set of genes works so that its presence in nature can be better appreciated, as well as to increase applications of bioluminescence as a tool into new fields and industries. In this experiment a library of genomic DNA will be created from V. fischeri in hopes of successfully cloning and expressing the bioluminescent phenotype into the DH5 α strain of Escherichia coli (this strain will be used because it has the ability to undergo high- efficiency transformation with foreign DNA). To do this, DNA was isolated and quantitated, digested with restriction enzymes, and will be ligated into a screening and expression vector and transformed into the recipient organism. Successful insertion of a fragment containing the lux operon can be identified by the color of a colony through antibiotic, blue/white and luminescence screening. The selected glowing clones from the genomic library will then be used for plasmid isolation through a series of mini-preps. Finally, isolated plasmid will be digested in order to better understand the nature of the inserted genomic DNA by electrophoresis. The pGEM-3Zf(+) TM plasmid was used as a vector because it enabled experimenters to replicate a large number of plasmid copies in E coli and because it contains the β- lactamase and lac Z α genes, which will provide ampicillin resistance as well as a useful way to differentiate between successful transformants and colonies lacking a chromosomal insert. More specifically, the lac Z α gene is also a multiple cloning site, so
Background image of page 2
Image of page 3
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 04/07/2009 for the course BIOCHEM 651 taught by Professor Weibel during the Spring '09 term at Wisconsin.

Page1 / 12

MBL1-4 - MBL 1-Cloning the lux Operon from Vibrio fischeri...

This preview shows document pages 1 - 3. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online