Question

11) What is meant by a control plate? What purpose does a control serve? Which plates are control plates in the lab?

12) If there are any genetically transformed bacterial cells, on which plate /plates would they most likely be located? Explain your predictions

13) Which plates should be compared to determine if any genetic transformation has occurred? Why?              

Answer 1

11) What is meant by a control plate?

An experiment consists of two set-ups namely,

an experimental set-up (test) and a control set-up. Both are identical in which every condition is the same except one.

The set-up in which the condition/factor under study is missing is called the control.

11. What purpose does a control serve? Which plates are control plates in the lab?

Control plate is used to compare experimental results obtained for the factor/condition under study. This can be further better understood with the help of transformation experiment set-up.

12) If there are any genetically transformed bacterial cells, on which plate /plates would they most likely be located? Explain your predictions

Transformation method has been of importance in genetic analysis since it helps to introduce plasmid DNA into recepient bacteria after certain genetic modifications carried out in lab.

Plasmid DNA to be used for transformation experiment should comprise of a marker gene which will help us determine whether transformation has been carried out or not.(eg. pQE-30 consisting ampicillin resistance marker gene)

Recepient bacterial strains used should be sensitive strain (eg. E.coli DH5 strain which is sensitive to ampicillin and recombination- deficient strain RecA strain).

Genetic modifications can be carried out by making the recepient bacterial cells competant by giving ice cold treatment of calcium chloride. the state of competance allows for the uptake of the plasmid DNA easily and results in the efficient cloning.

Steps of transformation:

Mix the competant recipient bacterial cells with the plasmid DNA and keep it on ice for 10 minutes.

Expose the cells to heat shock at 42 °C for 1-2 minutes.

Immediately keep it on ice-bath, so temperature reaches room temperature.

Add the growth medium used earlier to grow the recipient cells(eg. LB medium) and incubate for 24 hours. Transformed cells are now ready for the experiment. These are going to be used for the test experimental set-up.

For control, used the recipient cells which were not made competant and hence were not transformed.

Experimental set-up:

Test set-up: Spread plate the aliquot of the transformed cells prepared   (competant cells+ plasmid) on the medium containing ampicillin and incubate for 24 hours.

Control set-up: Spread plate the aliquot of the recipient cells (non-transformed) on the medium containing ampicillin and incubate for 24 hours.

15) Which plates should be compared to determine if any genetic transformation has occurred? Why?

For the results, the test set-up plate and the control plate will be compared for the growth of the recipient cells.

Control plate will show no growth of the recipient bacterial strain used since, it is sensitive to ampicillin antibiotic present in the medium.

Test plate will show growth of the transformed cells due to the presence of ampicillin resistance genes. Indicating successful transformation.

Note: If the test plate shows no growth it indicates that the transformation procedure was not carried out properly.

The comparision of results obtained on both the plates indicate that transformation of E.coli DH5 strain was carried out using plasmid with a marker of ampicillin resistance gene efficiently.