Plasmid Fusion

& Pcr Essay, Research Paper

Josh Hyman

Per. 5

Plasmid Fusion

&

PCR

The AMGEN Lab that we have been doing for the past two weeks consisted of

two parts; Plasmid Fusion and PCR. Each one is a complicated procedure of genetic

engineering, with our own cheek cells and E.Coli supplied by AMGEN. I will start by

explaining the Plasmid Fusion lab.

The Plasmid Fusion lab consisted of four major parts; plasmid digestion, gel

electrophoresis, restriction enzyme inactivation and ligation, and the final step, plating out.

But, before I get into that I should define some parts of the lab. The main pieces of genetic

information we will be working with are plasmids. Plasmids are gene sequences found in a

loop outside of the main chromosome. Their main purpose is to code enzymes that digest

antibiotic enzymes. Antibiotics are chemicals that kill bacteria or interfere with their

growth or metabolism. Cells that have antibiotic resistance have an advantage because

they are able to grow in places that other cells can not.

Our main purpose in this lab is to give an E.Coli cell immunity to the antibacterials,

Ampicillin and Chloramphenicol by genetically doctoring its plasmids. The first step in

doing this is to cut the plasmids so that we can ligate the new pieces on later. The DNA

will be cut once twice or not at all because the process does not work all of the time with

all DNA. The part that makes the resistance enzyme will be left in tact and separated into a

smaller section of DNA. We do this so that we can isolate the genome so that we can later

attach other sections of DNA to it. The next step involves checking to see if the

Restriction Enzymes did their job by a process called Electrophoresis. First we suspend

the DNA in a solution of Agarose. Then an electric is applied, one end is + and the other

– . DNA forms ions, like most substances that dissolve in water. It separates into H+ and

DNA-. The DNA will move toward the + end through the Agarose. Since the Agarose

will stop many of the bigger pieces but the pieces that were cut will pass through because

they are smaller. Using a chemical called Ethidium Bromide (EtBr) and UV light we can

see how far our DNA made the journey. The farther it went the smaller the pieces and the

smaller the pieces the better the Restriction Enzymes worked. The next step involves three

chemicals; the two separated plasmids and a T4 ligator. Mixing these three together

should form one final strand. The two pieces will be ligated to form the correct genome.

The next step is to put the new resistant strand of DNA back into the E.Coli. This process

involves mainly hot and cold water. The DNA will be put in a iced solution with the cells.

The cells should have small holes in them to let the plasmid back in. They will then be

shocked by putting them in a hot water bath for two minutes and then put back into the

ice. The shock should open up the cell long enough for the plasmids to get through. If the

plasmid makes it through, the cell should accept the new plasmid and start producing the

resistance enzyme to Chloraphenicol and Ampicillin. The next and final part of the

experiment is referred to as plating out. The process involves four petri dishes, each

divided in half. Each petri dish contains a different substance. There is a Luria Broth (LB)

plate( Luria Broth is a excellent nutrient source for the E.Coli), a Chloraphenicol

plate(CAM), an Ampicillin plate(AMP) and a Chloraphenicol and Ampicillin

plate(CAM+AMP). We spread our new E.Coli over each plate and let it stand for 36

hours. The following results took place.

LB AMP CAM CAM/AMP

growth = + + +- + +-

none = -

little = +-

What these results mean is the following: Since there is growth on the Luria Broth

solution the cells are still alive and are able to reproduce; Since only two very tiny colonies

grew on the AMP plate we can assume that only a very few gained the Ampicillin

resistance; Since there was growth all over the Chloraphenicol we can assume that the

cells have been altered and that they can now grow in a antibacterial environment that

would have once killed them; Since there was only very little growth on the Ampicillin and

Chloraphenicol plate I will have to assume that the plate had either too much Ampicillin or

that just the presence of it in cell growth will kill cells.

The PCR lab was the second lab we did. PCR which stands for Polymerase chain

reaction. The main purpose of this lab was to extract our own DNA from our cheek cells,

prepare in for the PCR and then put it in the PCR machine. What PCR does and allows us

to do is make indefinite copies of DNA. Not just the whole strand, nature does that for us,

but we can copy just part of the DNA for intense studies.

The first part of the lab is to extract our own cells for the project. We did this by

gargling with a saline solution for one minute while we bit our cheeks. I was incredibly

sick with a fever at the time so this might have affected my results. The next part is to get

the cells ready for the machine . To do this we mixed the cells in a micropipeter with a

substance called Chelex. We then mixed our buccal cells with two compounds called

Master Mix I and Master Mix II. Now is the most important part, the thermalcycler. The

thermalcycler s main purpose is to break down the DNA in to small parts. The hydrogen

bonds around the base pairs break apart and the DNA is suspended in the solution. The

next and final step in the lab is to run a gel electrophoresis on the sample. After adding the

loading buffer you can run the gel.

The expected results should be that the well in which we loaded the solution into is

now a bright glowing color and the band start bright and eventually fade into black. What

the lab actually tested was the presence of a genome called tPA-25-ALU. tPA-25-ALU is

a gene inside the tPA gene(which everyone has a copy of) which is not found in all people

since it is an inherited trait. We have either 2,1 or 0 copies of it in our body. The reason

that that specific part of the gene was targeted was the different Primers we used. The

primer selects the certain gene in want to copy by it s nucleotide sequence. It then saves

that part and replicates it because it is the only strand in the entire PCR mix to reproduce.

I think that the fact that the wells were glowing means that the PCR did work and that the

glowing product in the wells is the product of the PCR experiment. This is because in the

PCR machine all the DNA is separated and jumbled, the primer helps to form the only full

DNA structure so when we added the yellow loading buffer it could only bond with the

full DNA which was the tPA-25-ALU.