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What is a Plasmid?

 A plasmid is a DNA molecule that is separate from chromosomal DNA which is


in double chain strands. Plasmids are circular DNA strands.

To the left is a Cell. The red


strands are Chromosomal DNA
and the Blue are Plasmids.
Note they are circular and
separate.

In our study we start with a plasmid that has the DDR1-YFP part. We want our end
product to be DDR2 – YFP. We want to remove the DDR1 part and insert DDR2. This
is done for scientific reasons. This is the plasmid you start with. It has 2 parts to
make it simplified to understand. One is the DDR1 and the other is YFP, as labeled.

The next step is to cut the DDR1 portion out of your plasmid. The process how this
was cut will be explained in detail later.

The same process was repeated to obtain a DDR2 part.


So in the end this is what you are doing:-

Now I will explain the cutting process. The process of how DDR1 is cut out from the
DDR1-YFP plasmid. You start off with the DDR1-YFP plasmid as show in the figure
below.

You place the plasmid in a buffer solution that contains restriction enzymes. In the
lab you can buy restriction enzymes from a company. You find out from scientific
journals which restriction enzyme your plasmid needs. In our case it needed 2
restriction enzymes to cut out the YFP, namely NheI and EcoRI. (You do not need to
know the names of the restriction enzymes).
What is a restriction Enzyme?

 They are an enzyme that cuts out a particular DNA molecule. Remember
plasmids are DNA molecules organized as a circle.

After you finish the cutting part, you have to freeze the entire solution. Why do you
freeze it? You freeze it so you make the restriction enzymes inactive otherwise they
will cut up the rest of the molecules. These reactions usually take place at 37
degrees Celsius because all DNA molecules are active at that temperature. Freezing
is usually carried out at -20 degrees Celsius.

Now since you are interested in having the YFP (Yellow part in figure above), the
next question is how do you separate it from the rest of the junk you do not want.

The answer is GEL ELECTROPHORESIS. It is a common molecular biology


technique used to separate different DNA segments and molecules.

Your sample is in a vial like


the blue vial to the left. It
contains the fragments
(DDR1, YFP, Restriction
Enzyme 1 and 2).

The liquid is then injected


into wells in the grey tray to
the left. I will explain the
equipment set up in the next
part.

Now DNA has a negative


charge.

Read points 2, 3 and 4.


Gel Electrophoresis - Process used to separate DNA molecules, proteins, etc. The
process works on the principle of separation by charge and size.

Equipment looks like this: Animated Version Looks like:

The concept is to separate molecules based on size and use the characteristic of
charge to separate it. The equipment consists of GEL, a cathode and an anode. The
cathode (-ve) charge end is where the sample is inserted. When the power is turned
on the molecules move towards the anode (+ve) charge. The bigger/longer
molecules move slowly and the smaller/shorter molecules move faster. When you
insert your sample you put in a dye which allows you to see the movement of your
molecules. You can visualize this with image below.

This is how much you should now regarding the concept. When you are working in
the lab you also have to make the gel. The gel is a sponge like texture. It has holes
in it. So when the sponge has smaller holes it is hard for the bigger/longer
molecules to pass through it hence allowing the shorter molecules to move faster.
Remember ALL biological molecules have a negative charge. So movement will
always be from cathode (-ve) to anode (+ve).

A very good animation to understand GEL electrophoresis is at this link:

Please read the text and look at the animation at each stage to understand it.
http://www.dnalc.org/resources/animations/gelelectrophoresis.html

At the end of GEL ELECTROPHORESIS, your GEL looks like this:

Each band is a molecule or a fragment that was in the liquid you wanted to
separate. So one of the bands would contain the YFP product.

Then you use a special EXTRACTION Kit to purify or remove the gel to obtain your
part of the YFP. There are references you use to know which part of the gel you
want or has your part. Each band is a specific size, you know this from references.

Now what you have is a few pieces of YFP. Remember that you are working with
molecules the size you cannot see. So now you have to increase the number of
YFP’s you have. The term to use, is amplify.

If you only have one piece, you are mostly likely going to damage it. So you need to
amplify or multiply the number of pieces. To amplify or increase the number of
YFP’s you have you use a process called PCR. Polymerase Chain Reaction.
What is Polymerase Chain Reaction (PCR)?

 It is a scientific technique used to amplify (increase or multiply) a single or


few copies of a piece of DNA to generate several million copies of that
sequences.

 This process is done through a series of reactions in test tubes or vials, like
the one in the image below.

The PCR reaction takes place in 3 steps.

 You know that DNA is double stranded.

 So the first step is to heat the DNA up so it separates into single strands.
Heating at about 90°C will separate the strands.

 The next step is to lower the temperature to about 50°C so that short
stranded pieces of DNA called PRIMERS can attach to each chain.

Primers
 Now again you raise the temperature to abt 70°C and allow a special type of
DNA that is not affected by heat to extend the primer strands (red and blue
strands are extended).

 Now you see that you have 2 strands when you started off with one. This
process is repeated automatically millions of times in a PCR Machine
(automation) to produce the millions of copies in a very short time.

So ultimately you end up with several million copies of the YFP. You can gain a
better understanding through this step by step animation:-

http://www.bio-rad.com/flash/07-0335/07-0335_PCR.html

Remember your final product is DDR2-YFP. So you repeat the same steps you did
for YFP and obtain the several million copies of DDR2.

In the end you have millions of copies of DDR2 and YFP that are separate. So
how do you join them?

The process is to mix together the two different parts and put them into a cell
culture of bacterial cells. The cells will absorb these parts where they will join
inside the cell. Your part of the project STOPs here.

Bacterial Cell with many copies of DDR2-YFP


I will give you some background for the rest of the project.

Once you obtain millions of copies inside the bacterial cell, they are removed
from the cell through a process called transfection and inserted. Same way you
produced DDR2-YFP receptor, you will also produce DDR1-CFP receptor.

When you obtain millions of copies of each of these, you put them in a cell
culture with human cells where they attach to the surface as receptors.

Then you use microscopy called FRET analysis, to analyze how they function
when they are activated.

What you should have learnt in this part is details :-

- Details of PCR and GEL ELECTROPHORESIS.

- How you constructed a plasmid.

- Overall understanding of your part of the project.

After reading and understanding the above points you should be able to
understand the following points on your resume.

 Investigated the activated state of a receptor on mammalian


cell surface. [OVERALL OBJECTIVE]

 Constructed plasmids that expressed the desired DNA and


protein sequence. [Described in process above]

 Gained experience with molecular biology techniques such as


PCR and Gel Electrophoresis. [Also explained in prior pages]

 Gained knowledge pertaining to transfection and Fluorescence


Resonance Energy Transfer Analysis (FRET). [Explained on last
page, last paragraph]