Promoters

- If we want an inserted gene to be expressed in a bacterium, a promoter must be added.

- In the case of insulin, the insulin gene was inserted next to the beta-galactosidase gene
(controlled by the lac operon), so they share a promoter. This means that if the bacteria are
grown in a medium containing lactose but no glucose, they would synthesise both beta-
galactosidase and insulin.
- The promoter allows RNA polymerase to bind to the DNA and identify the template strand of the
two antiparallel DNA strands.
- In eukaryotes, transcription factors may also need to bind to the promoter or RNA polymerase
before transcription can occur.

Electrophoresis
- Electrophoresis is used to separate individual charged molecules or ionic substances in a
sample on the basis of their differential movement in an electric field.
- The essential components of an electrophoretic system are:
Two electrodes, the anode (positive) and the cathode (negative) between which a voltage is
applied
A buffer solution through which the current flows and in which the ionic substances move at
constant pH.
A supporting medium which limits diffusion.
- The medium for gel electrophoresis is polyacrylamide (for proteins) or agarose (DNA).
- The movements of the charged molecules in the gel depend on:
The net charge: Negatively charged molecules move towards the anode and positively
charged molecules move towards the cathode. Highly charged molecules move faster
then those with less overall charge.
Size: The smaller the molecules, the faster they move.
Composition of the gel: The size of the pores within the gel determines the speed at
which proteins and DNA fragments move.

Steps for the Electrophoresis of Proteins

1. The protein sample is mixed with a buffer solution, a substance to cleave disulfide bonds, and
a tracker dye. Then, it is heated to linearise the polypeptide chains.
2. Individual samples are added to the wells using a pipette.
3. The gel (polyacrylamide) is positioned with the wells closest to the cathode. Most proteins have
a net negative charge. Apply the voltage.
4. Fix and stain the gel.
Separated proteins are visible as bands.
5. Examine the results. The position of the bands can be compared with molecular mass
standards to determine the size of each band.

- Amino acids have R groups with different charges (e.g. -COO - and -NH3 +). This allows them to
undergo electrophoresis.
- The haemoglobin of people who have sickle cell anaemia will have a less negative charge
because valine is replaced by glutamic acid. So, the molecules of haemoglobin with the
defective beta-goblin chain will not move as far through the gel as those normal ones.Thus, the
sickle cell allele can be checked for with electrophoresis.

Steps for the Electrophoresis of DNA

1. Prepare the gel (agarose).
2. Prepare the samples and add a tracking dye.
3. Load the samples onto the gel.
4. Load the DNA markers: these standards are of known size and are added to the first and last
wells of gel.
After electrophoresis the relative position of the bands of known size can be used to prepare a
calibration curve.
5. Carry out electrophoresis for 30-60 minutes until the tracking dye has migrated across 80% of
the gel.
6. Examine the results by transferring the gel to a UV transilluminator.
7. Extract only DNA bands of interest.

- DNA fragments are negatively charged due to the charged phosphate groups attached to them.
Thus, the DNA fragments move towards the anode.