Question

PLATING AND GROWTH OF TRANSFORMED CELLS:

3a. Some plates will have LB mixed with ampicillin on them. What is ampicillin and what does it “normally” do (when not used in the lab)? How (molecular mechanism)? Does putting ampicillin in the growth media allow scientists to determine whether bacteria took up the pAMP plasmid, the Kan^r-pAMP recombinant plasmid, and/or something else? Explain your answer.

3b. Some plates will LB mixed with kanamycin on them. What is kanamycin and what does it “normally” do (when not used in the lab)? How (molecular mechanism)? Does putting kanamycin in the growth media allow scientists to determine whether bacteria took up the pAMP plasmid, the Kan^r-pAMP recombinant plasmid, and/or something else? Explain your answer.

3c. Why is the efficiency of transformation of recombinant plasmid so low?

3d. Besides accepting a recombinant plasmid, what are three alternate ways bacteria could grow on plates with ampicillin and kanamycin? (clues: natural ways, other transformed product, faulty lab reagent(s))

Answer 1

3a. Ampicillin is an antibiotic, working against both gram+ve and gram -ve bacteria. It is normally used to treat various bacterial infections like respiratory infection, meningitis salmonellosis and many more.

Molecular mechanism: The ampicillin can penetrate through gram-positive and some gram-negative bacterias, it contains an amino group which helps it pass through the small pores in the outer membrane of gram-negative bacteria like E.coli.

Putting ampicillin will help the scientists to determine whether the bacterial colony have taken up pAMP plasmid, the pAMP plasmid contains an ampicillin resistance gene, if the colony takes up the plasmid, it will survive even when there is ampicillin added.

3b. Kanamycin is an antibiotic also called aminoglycoside bacteriocidal antibiotic, generally used to treat many bacterial infections when not used in the lab,

Molecular mechanism: It works by interfering with protein synthesis in the bacteria, the kanamycin binds to the 30s subunit of the bacterial ribosome, which leads to the incorrect formation of mRNA eventually leading misread causing wrong amino acid in the peptide.

The bacterial colony will take up the kanr-pAMP plasmid, even though kanamycin is used, as the kanr-pAMP gene helps to resist the antibiotic added. As the plasmid contains a kanamycin-resistant gene that is said to be resistant to the antibiotic ad when bacterial colony takes up the Kanr-pAmp gene, it will fruitfully grow in the media added with kanamycin.

3c. The factors affecting transformation efficiency, are as follows:

• Plasmid size: A big plasmid molecule has low transformation efficiency, as the size of the plasmid increase, the efficiency decreases.
• Forms of DNA: A highly coiled DNA molecule (plasmid) finds it difficult to pass through the small pore created on the cellular membrane of the bacteria.
• Methods of transformation: The method of preparing competent cells, plays a major role. For example, when the heat shock method is used, the exposure of bacterial colony to extreme temperature for a prolonged duration of time will lead to rupture of the cell and death of many bacterial cells in the colony, which indirectly will decrease the transformation efficiency.

3d. Besides accepting a recombinant plasmid, the three alternate ways bacteria could grow in plates with ampicillin and kanamycin are:

• natural ways: Mutation, this is a very natural phenomenon happening in living beings like bacteria whose reproductive rate is high, this makes them more prone to mutation and by some mutation, they might acquire resistance against any of the two antibiotics.
• Other transformed product: The mutated bacteria (containing antibiotic resistance gene), may transfer their mutated DNA to other bacteria, this mechanism may also lead to obtaining antibiotic resistance by the bacterial cell even if transformation does not take place.
• Faulty lab reagents: Antibiotics like Ampicillin and kanamycin are basically protein molecules, which are prone to get denatured when subjected to hot temperature or not stored in a cool and dark place. Using these kinds of reagents will give a false-positive result in the experiment.