SECTION A
1. C
2. A
3. B
4. C
5. C
6. D
7. A
8. C
9. C
10. B
11. D
12. A
13. C
14. D
SECTION B
1. Predicted size of protein = number of amino acids  0.11kDa
=
1130
3 amino acids  0.11kDa
= 41.43 kDa
 41 kDa
2. Four phases of bacterial growth curve.
(i)
Lag phase
(ii)
Log/exponential growth phase
(iii)
Stationary phase
(iv)
Death/Decline phase
Figure 1: Diagram representing bacterial growth curve.
3. (a) When the cell density is 0,6-0,8 the bacteria are usually in the exponential growth phase
and at this point the entire cell is wired for growth.
(b) Addition of IPTG helps to bind to any repressors in the operator region of lac operon. When
this happens, RNA polymerase finds its way to synthesize RNA molecules which later be
translated to protein. This process only occurs in the presence of sugar. Without IPTG,
repressors stick to the operator region and inhibit RNA polymerase to start synthesizing RNA
molecule, as a result, no protein is produced or the process can occur slowly.
4.
(a)
Components
Coomassie Brilliant blue
Function
It is used as a common analytical dye in SDSPAGE.
sodium dodecyl sulfate (SDS)
It is used to denature proteins.
Glycerol
It is used to add density to the sample.
β-mercaptoethanol
It is used to break all the disulfide bonds.
(b) What do you need to do to in order to analyse the gel?
•
The gel must be stained using a staining buffer.
Components of the staining buffers
(i)
Coomassie Brilliant blue
(ii)
Ethanol
(iii)
Destilled water
(c)
Discontinuous gel electrophoresis consists of staking and resolving gel with different
concentration gradient of polyacrylamide. As a result, proteins separate better as they migrate
through different gradient producing high separation resolution of different proteins.
5. Affinity chromatography is a separation method based on a specific binding interaction
between an immobilized ligand (Nickel beads) and its binding partner (His-Tagged protein).
In this technique, the stationary of this chromatogram is made up of beads that has high binding
affinity to the histidine tagged proteins. As a result, proteins with the tag binds to the beads
and the remaining molecules with no tag stays unbound. Furthermore, the use of imidazole
enables to elute the protein of interest since it has higher binding affinity as compared to the
histidine tag in protein of interest.
(i)
Binding:
A sample of different proteins can be used in order to purify only protein of interest.
However, only his-tagged protein will bind to the nickel beads since it has high binding
affinity. The rest will not bind to the beads as indicated by figure 2 (i).
(ii)
Wash
To remove the unbound molecules or proteins, the washing step is applied. The
washing buffer used to wash unbound molecules consists of low concentration of
imidazole. The protein of interest will remain bound to the beads since it has high
binding affinity through the tag as indicated by figure 2 (ii).
(iii)
Elute
In order to elute the protein of interest, high concentration of imidazole is used as elution buffer
to remove the bound proteins since high concentration of imidazole have more binding affinity
to the beads than his tagged protein. As a result, protein of interest in then eluted as indicated
by figure 2 (iii).
(i)
Bind
(ii)
Wash
(iii)
Elute
Figure 2: Diagram representing Nickel affinity chromatography.
6.
Marker
Gel
Dimer (82 kDa)
Figure 3: Diagram representing a formation of dimer