Microsoft word - lab110804b.doc

BIOLOGY DCN
TRANSFORMATION of E. coli LAB #11
This is a relative term. The more colonies on the plate the more successful the transformation. Use the equation in the manual to calculate your own transformation efficiency. 2. How did each of the controls validate the experiment? • the cells used for the transformation were healthy • X-GAL had no obvious detrimental effects on the growth • X-GAL was not being hydrolyzed (to blue) by any Control #2: plate with ampicillin and X-GAL No growth on this plate indicated that the ampicillin was effectively killing the cells Growth would have indicated that the ampicillin was not working, either because its effectiveness had expired or when making the medium, the ampicillin had been added when the medium was too hot, which would have destroyed the ampicillin 1. Did you observe any satellite colonies? Why are satellite colonies white?
Following a 24-hour incubation, no satellite colonies were observed
Following an additional 24-hour incubation, satellite colonies were
observed.
Satellite colonies are white because they cannot hydrolyse X-GAL to yield
a blue product. These are cells in which the lacZ gene in the bacterial
chromosome contains a deletion that renders the protein (β-galactosidase)
ineffective. Each satellite colony began as a single bacterial cell that had
not been transformed by the plasmid. The cell was initially able to survive
in the medium, but was not able to grow and divide because cell wall
synthesis was inhibited by ampicillin present in the medium. Once a
nearby transformed cell developed into a colony by many divisions, the β
–lactamase produced (the ampr gene was transcribed and translated to
generate β–lactamase) by the colony diffused into the medium. Once in
the medium supporting the non-transformed cells, β-lactamase inactivated
the ampicillin, making cell wall synthesis and cell division by non-
transformed cells possible.
2. Why did the competent cells, which did not receive DNA (control), fail to grow These cells did not contain the plasmid with the ampr gene, so cell wall synthesis was inhibited and the cells eventually died. 3. How could a cell, which has been transformed by intact pGAL DNA, give rise to a white colony on a plate containing AMP/X-GAL? A cell could have given rise to a white colony due to a mutation in either part of the lacZ gene present in the bacterial chromosome or in the plasmid. In the case (we didn’t do this in the lab) where there had been an attempt to insert a piece of foreign DNA into the truncated lacZ gene (which contains the multiple cloning site) in the plasmid, it is possible that the plasmid could have reconnected (without the insert) in a way which deleted a base (or two) from the truncated lacZ gene. In this case, the truncated lacZ gene would have been transcribed and then translated out of the correct reading frame, rendering the product unable to complement the modified lacZ gene product of the chromosome. 4. Why are there so many cells growing on the X-GAL plate? What colour are There are many cells growing the X-GAL plate because there was no ampicillin in the medium, so cell wall synthesis and therefore cell division could proceed. The colonies were white because they did not contain the plasmid (they were not incubated with pGAL to begin with), which provided the truncated lacZ gene. 5. The antibiotics, kanamycin and tetracycline, interfere with protein synthesis. Competent bacterial cells can be transformed with plasmids encoding resistance to these drugs. When the cells are plated on agar medium containing these antibiotics, satellite colonies are not found. Increasing incubation times and the amount of cells plated do not give rise to satellites in the case. Why? Kanamycin and tetracycline both function inside the cell. The resistance proteins, i.e. the ones that degrade these antibiotics, are not extruded from the cell like β–lactamase is. Since it is not extruded, the region surrounding the cells has not been rendered “antibiotic free” by the resistance proteins, so untransformed cells are unable to grow. The untransformed cells that landed in the region were killed relatively quickly, since protein synthesis was interrupted soon after exposure to the antibiotics.

Source: http://jacusers.johnabbott.qc.ca/~biology/DCN/labs/lab11.pdf