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Inserting jellyfish DNA into bacteria


Earlier this month, we ran a one-day course on one of the most essential techniques in biotechnology: transformation. It is a practical tool in the lab for making many copies of your favorite gene by introducing it into bacterial cells like E. coli and it is actually a very straightforward approach that some scientists use nearly every day. The main requirement is that the bacteria are in a state of competence which allows one to trick the bacteria into taking up foreign DNA. There are different ways of transforming bacteria, but for our course we used the classic heat-shock method. The bulk of the equipment we used included a bucket of ice, microwaved water, and an antique incubator to grow the cells in.

Essentially, we made in-house competent E. coli by gently bathing them in calcium chloride on ice. Next we added the DNA and heat shocked the poor little bacteria. I sympathize with them because upon heat shock most of them die, but the bacteria that do survive have a chance of taking up the DNA. The sudden shock of high temperature makes their cellular membranes more porous which allows the DNA to enter them. After the shock, we spread them on agar plates containing antibiotics or other chemicals to select for bacteria that actually took up the desired DNA. If foreign DNA is successfully introduced into bacteria it will be replicated while the bacteria divides, and you get many copies of the DNA.

We transformed two types of DNA which we ordered through Carolina Biological: pGREEN and pBLU. Both of these came in the form of double-stranded DNA, known as plasmids. There are many essential components within these plasmids but the noticeably important ones are the genes of interest and the antibiotic resistance genes. The pGREEN plasmid contains the gene encoding Green Fluorescent Protein (GFP), which comes from our jellyfish friends. GFP glows a mesmerizing neon green when illuminated under blue or UV light. The pBLU plasmid contains a gene called LacZ which encodes beta-galactosidase which when exposed to a drug called X-gal, produces a blue pigment. Finally, both plasmids contain an ampicillin resistance gene which allows bacteria which take up these plasmids to survive on plates treated with the antibiotic.

After the plates were given a couple of days to grow, most of our students got plates full of green glowing bacterial colonies! To detect the GFP fluorescence, Dennis brought in a compact UV flashlight and revealed the brilliance of the GFP colonies. The pBLU plasmid proved more difficult to transform, but one of our students managed to get some blue colonies! We had an awesome group of participants and had a blast running the course. This class is just some of the progress that we have made in BosLab this year, we plan to hold more events and start new projects next year. In January we are leading a local study group for the EdX Synthetic Biology course taught by Ron Weiss at MIT. Look out for upcoming events in 2016!

For more information, check out our Molecular Biology 101 manual.

More photos from the class:

Carefully adding DNA to the competent bacteria.

At the lab bench!

Raphael prepping everyone for the transformation protocol.

pGREEN transformed colonies glow green.

pBLU transformed colonies.

#Ecoli #course #GFP

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