-You have three separate plasmids in your lab, but the labels have come off of t
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Question
-You have three separate plasmids in your lab, but the labels have come off of the tubes and you no longer know which plasmid is which. For the time being, you will refer to the plasmids are plasmids 1, 2, and 3. You know the following:
-One plasmid has kanamycin-resistant gene.
-Two plasmids have an ampicillin-resistance gene.
-In addition to having an antibiotic-resistance marker, one plasmid also codes for the gene for green fluorescent protein (GFP), a protein from the bioluminescent jellyfish Aequorea victoria. Bacteria that produce the green fluorescent protein look green under white light and fluoresce under ultraviolet light.
-Your PI has tasked you with determining the identity of the antibiotic resistance gene in the plasmid containing GFP. Write a detailed protocol describing exactly how you would do this. Include all steps of the transformation making sure to include all the controls you will need. Your protocol should be clear enough for a brand new student in your lab to follow.
Explanation / Answer
Answer:
Given here is that we have 3 plasmids.
Out of three, 1 has kanamycin resistat gene , and 2 has ampicillin resistant gene
Also, any one of the plasmid has GFP protein which glows in UV light.
We need to know the plasmid which has GFP and also what antibiotic resistance it has (either kanamycin or ampicillin ).
Therefore,
For the transformation step,
Transformation is done. And now for plating, we plate each plasmid cultures in LB plates for checking the production of GFP. Also one control plate is required where no plating should be done.
The cultures are plated and labelled as 1, 2 and 3.
After overnight incubation the growth was checked under UV light . Now lets say for example plate 2 i.e plasmid 2 was showing the GFP production.
Now we need to know which antibiotic resistance it is showing. For that we take a loopfull of culture from plate 2 and steak into two plates, one containing kanamycin + LB and the other containing ampicillin + LB . And ofcourse 1 control plate with no streaking.
Now lets say the kanamycin plate showed growth after overnight incubation and this plate, when exposed to UV glowed. Thus, this proves that the plasmid containing GFP contains kanamycin resistant gene.
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