Inside Room 808 on a crisp April morning, Class 12 students were seated at lab tables with an array of scientific tools in front of them. “Take your protocol out please!” announced a visitor at the front of the room.
Their guest, Jenny Hackett, Ph.D., was a familiar face for these students. A member of Cold Spring Harbor Laboratory, Dr. Hackett had previously visited Chapin to commence a three-part Mutagenesis (the process of manipulating genetic information) Lab with the seniors in our Molecular Genetics course. On this particular day, she was ready to help them complete it.
The highly advanced lab—of Dr. Hackett’s design—was a prime example of genetic engineering in action as our seniors were taking a green fluorescent protein gene (GFPuv)—originally from jellyfish—and mutating it to make it glow in a color of their choice.
In earlier sessions with Dr. Hackett, the students were given a chunk of the gene and asked to locate the correct sequence for mutating. From there, they each mutated an amino acid (or two, if they were changing to yellow) in the DNA green bioluminescent gene to make bioluminescent cyan, yellow or red. Using DNA primers of their own creation, the students isolated the gene to conduct a PCR (Polymerase Chain Reaction), thus producing millions of copies of the gene, and performed gel electrophoresis to ensure their DNA wasn’t destroyed when altered.
Today, they’d finalize their mutation by combining DNA fragments and inserting them into bacteria. “Set your pipettes to the correct microliters,” Dr. Hackett instructed. “Make sure to label your tubes,” she reminded the students as they worked to mix their fragments with a special enzyme. “The clear tube should have no exonuclease and the purple should read plus exonuclease.”
After observing Dr. Hackett perform the necessary steps, the seniors transferred their own assembly tubes to a 30°C water bath and obtained a separate tube containing 15 μL of Stop Solution, which takes away the magnesium ions that the enzyme needs to work. After timing exactly 10 minutes, they added 5 μL of Stop Solution to the purple tube with exonuclease, pipetting up and down to mix, followed by 5 μL to the tube without exonuclease. Those tubes were then placed on ice until ready to transform.
“We’re working with ‘competent bacteria,’” Dr. Hackett explained, which means “they’re treated with chemicals so they’re ready to absorb DNA.”
Next, it was time to heat shock the tubes for exactly 90 seconds to ensure the DNA could slip inside. “Timing is super important here,” Dr. Hackett emphasized, noting that the tubes would immediately return to ice afterwards.
Only the bacteria that successfully took in the students’ DNA will grow—ignoring universal bacteria that could contaminate the specimen—because the 12th grade scientists ensured their mutation included ampicillin resistance.
“It really is amazing what these students are doing,” remarked Science Teacher Jill Hirsch, noting that this is a “faster and fancier” way for scientists and researchers to conduct mutagenesis. As the period came close to its end, Dr. Hackett again reminded students of the importance of labeling, saying, “Ensure your bacterial plate has your initials, the date and plus and minus exonuclease.”
After a 48-hour incubation period, the students returned to Room 808 to view their bacterial plates under a UV light and, to their delight, saw them successfully glow in a new color!
