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Information presentation Molecular Biology Experimenting (MBE)
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Information presentation Molecular Biology Experimenting (MBE)
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Electroporation / transformation The purpose of electroporation and transformation
During MBE, transformation is used to post a plasmid with the gene of interest in a bacterial
cell. This is done in order to be able to do further research with the plasmid, where the gen
of interest is inserted. We can also make more of the plasmid with this method. The
electroporation in a eukaryotic cell is carried out by placing the gene of interest in a Dicty
cell, by means of a plasmid. This is done to let the gene be expressed, this is used for
example to localize the gene in a cell.
Method and technic
Transformation is a genetical alteration of a cell, which is the result of the uptake of
exogenous genetic material from its environment through the cell membrane. Bacterial cells
and eukaryotic cells do not just take up foreign DNA, you need to do chemical
transformation or electroporation to make competent cells. Competent cells are cells that
can take up foreign DNA. The chemical transformation is used by E.coli cells, this is also
called heat shock or the calcium chloride method. In chemical transformation the cell wall of
bacteria can be made weak by incubate them at a low temperature in a buffer with certain
salts. When you add the plasmid to the cells, and incubate them short on a high
temperature, a portion of the DNA will be added in bacteria. Electroporation can be used for
bacterial cells and eukaryotic cells, when they are exposed to an electrical field the
permeability from the cell membrane will be higher. This is the reason why foreign DNA can
be introduced in the cells.
Results and conclusion
There is a change that treated bacteria are died during the transformation. And what
method you use, there will always be many cells who haven’t taken the added plasmid. To
keep only the good bacteria, we use a selective culture medium. In this case, we added
Ampicillin, when the bacteria don’t make a protein which is resistant to ampicillin, they will
die. On agar plate 2 growth is present, this means that the plasmid is incorporated. Because
the cells have become resistant to ampicillin. On agar plate 6, the negative control, there is
only E.coli present, so these cells have to die. But, on our plate are colony’s present so this
means that something went wrong. There was a lack of ampicillin added, and this wasn’t
good divided on the plate. The conclusion is that our transformation has gone well, because
if you have growth on an Ampicillin plate then you can be sure that the plasmid is
incorporated. But the negative control makes the results less representative.
These are the results from the electroporation, on this picture you see the positive control.
We did a positive control to confirm that electroporation doesn’t kill the Dicty cells. On the
other picture, are the electroporated cells with DNA, you see a big clump of cells.
, IC 50 determination
The purpose of the IC50 determination
During MBE, the purpose of the IC50 determination is to determine the effect
of kojic acid on tyrosinase. The effect of kojic acid on tyrosinase is determined
by looking at the amount of kojic acid when the tyrosinase activity is 50 %
inhibited.
Method and technic
IC50 is a quantitative measure that indicates how much of an inhibitor is
needed to inhibit a biological component by 50 percent. An enzyme, cell or
micro-organism could be the biological component. In this case the biological
component is the enzyme tyrosinase and the inhibitor is kojic acid. Inhibitors
cause the inhibition of the conversion of the substrate. There are two different
inhibitors : the competitive inhibitor and the non-competitive inhibitor. The
inhibitory effect is caused by kojic acid that forms complexes with copper.
Tyrosinase is containing a metal ion, as a cofactor. This means that kojic acid
will form complexes with tyrosinase too, this causes the inhibition of the
conversion of L-dopa. L-dopa provides enzyme activity, this enzyme activity will
be less. When we do this research, as first the protein will be isolated. The
optimum wavelength will be determined from this protein. Also the optimum
amount from the enzyme and the substrate will be determined. With this
information the inhibition by kojic acid will be determined.
Results and conclusion
In this graph you see the percentage enzyme activity and the amount of added
kojic acid. Now, you can determine the IC 50 value from tyrosinase, that is
upon the addition of 7 microliter kojic acid. You can read it in the graph by
taking y= 50. This is the time in which the reaction is 50 % inhibited.
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