Question

1) You have cloned the human Insulin (Ins) gene into the pDrive plasmid, and transformed bacteria using (0.25 ug) of this pDrive/Ins (as per the protocol described in this lab). Using the information below, calculate the number of molecules of Insulin that have been cloned into your entire culture after 4 hours of growth after incubation.

a. The transformation efficiency of this reaction is 10⁵ colonies/ µg intact pDrive/Ins plasmid.

b. pDrive plasmid has a copy number of 100 molecules per transformed cell.

c. Following the heat shock the entire 300 µl of cell suspension was used to inculcate a 20 ml of fresh LB broth.

d. Transformed cells enter lag phase 40 min after inoculation and begin to replicate on average every 20 min.

Answer 1

SOLUTION:-

The number of bacteia that get transformed :-

= Amount of DNA transformed x transformation efficiency

= 0.25 x 105

= 25000

• Since complete 300 ML of the transformed sample was plated, the number of bacteria in culture = 25000
• Number of generation of bacteria = ( total time of incubation - log phase period)/division time

Number of bacteria after 9 generations :-

= (2 ^ 9)x 25000

= 12800000

Number of insulin clones per cell = 100

[ because of the copy number of the plasmib ]

Number of insulin molecules in culture = 12800000 x 100

= 1280000000

Therefore, number of insulin molecules in the culture after 4 hrs = 1.28 x 109

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