BIOL1181 2250 Assignment 1: Applied Theory (WEEKS 1-3)

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Question 1. You have recently joined a biotech company that specialises in the cloning and expression of human

hormones for therapeutic use [Total = 12 marks].

Your first task it to express the Human Growth Hormone, whose structure is depicted below:

Genomic DNA 5

5'

size (bp)

cDNA

GH protein

BIOL1181 Assignment 1: Applied Theory (WEEKS 1-3)

Total Marks = 35

nukleotides 5175 5234

I

exons

Answer:

▼

IVS 1

Answer:

10 261

5'NT

5495

|

✓

161

5655 5865 5984 6077

|

✓

✓

IVS 2

✓

IVS 3

209 120 93

31 40 55

IV

6241

165

•

65

IVS 4

253

6495

1

▼

-3-23 1-31 32-71 72-126 127-191 (aa)

V

195

6689 (21)

You need to provide your supervisor with a strategy to clone this gene in an expression vector for expression in a

bacterial cell system. You need to:

3'NT Poly A

a. Explain to your supervisor which starting material you will use (genomic DNA? mRNA?) to prepare

your insert, and why? [2 marks].

b. List the elements that must be present in this vector for the successful expression of the bacterial

system, and why these elements are required? [4 marks].

BIOL1181 2250 Assignment 1: Applied Theory (WEEKS 1-3)

c. List the mains experimental steps required for cloning your insert of interest into the vector, starting

from extraction of the relevant nucleic acid from cells (as you have defined under Question 1a) and

finishing with a pure population of bacteria that has acquired the plasmid. Indicate which enzyme(s)

is(are) are required at each step [6 marks]? You can use a schematic.

Answer: