PGlo Transformation

Purpose

The purpose of this experiment was to test the idea that cells can take in genetic information. By using the glo gene, and putting these cells in different situations we were able to see what it takes for a cell to incorporate foreign DNA into their own DNA. This also helped us learn how viruses are able to take over an organism.  

Introduction 

In this experiment we did genetic transformation. Genes contain pieces of DNA that provides the information to make the proteins. Proteins gives an organism their trait. When changed caused by genes it is consider genetic transformation and many use in science. One application would be biotechnology in the fields of agriculture and medicine. For example, with bacteria by moving genes with the help of the plasmid, which the circular DNA. Plasmid DNA usually contains more than one trait that helps the survival of bacteria. Bacteria can transfer plasmids to adapt to new environment. Bacteria becomes resistance to antibiotics because of this transfer. Green fluorescent protein or GFP can by transformed by plasmids. It causes them to glow. When the transformation is done the bacteria will glow fluorescent in the dark. The PGLO plasmid codes for this gene that is resistant to antibiotic ampicillin. To control the rate of proteins that transferred into a cell caused by gene regulation system. Sugar arabinose can be active by GFP. This procedure takes place on antibiotic growth plate

Methods

First, we put in transformation liquid into
each tube; one labeled +pGlo and another
-pGlo.

Then we put them on ice .

Next, we transfered e coli to the tubes. 

We added plasmid DNA only to the
+pGlo tube.

Then we cold shocked the tubes for
10 minutes.

While we waited we labeled our petri dishes

Next we heat shocked the cells by putting
the tubes in a water bath, and back again into
the ice.

Then we added LB nutrient broth to the
mixtures, and mixed.

Lastly, we spreaded the solutions onto
the petri dishes, and let them
incubate for one day. 

Data and Graphs:

Discussion:

The LB/-PGLO plate had the most bacteria that resembled the original untransformed E. coli colonies we initially observed. This makes sense, if we take into consideration the fact that the bacteria were removed from the starter plate, did not have any plasmid added to them, and only had LB (this is broth, or in simpler terms, food) on the plate. Regulating these features makes it a control plate. The other control plate is the LB/amp/-PGLO plate, which had zero growth, because no plasmid was added. Plasmid actually has to be added in order for bacteria to multiply in the presence of ampicillin. The transformation plates include LB/amp/+PGLO and LB/amp/ara/+PGLO. First, both plates had the plasmid for the fluorescent gene added to the E. Coli spread on them. Because of the heat shock we gave the E. Coli, holes could be made in the cell membrane of the prokaryote cells. This means that the PGLO plasmid which expresses the ampicillin resistance gene is incorporated into the E. Coli. Thus the bacteria can survive on the plates that contain ampicillin. On the other hand, cells that were not treated with DNA did not express ampicillin resistance and did not grow on the LB/amp/-PGLO plate. The LB/amp/ara/+PGLO plate had about the same amount of growth as the other transformation plate, but the genes of the plasmid will only be expressed in the presence of arabinose, so it’s on this plate that the bacteria glowed under a UV light. The transformation efficiency (efficiency by which cells take in extracellular DNA and express its genes) for this plate is 406.25 transformants/microgram. This basically reflects how competent prokaryotic cells are at including new DNA. Our results are what they were supposed to be, and they support our belief that only the cells with DNA added to them would be genetically transformed. One way to improve this lab could be to let it go on for more than one day. That way we would try to calculate rate of reproduction in order to grasp how quickly plasmids can be generated in new cells.

Conclusion :

 We found that we were able to transform the DNA of this organism using plasmids. We concluded that E. Coli did not grow. Some bacteria was able to live in the surroundings with ampicillin  and glow under UV light in a environment with arabinose. Based on these  result our  hypothesis came true. 

Gel Electophoresis Lab

Background Information:

Gel Electrophoresis is a type of DNA technology that's used to separate nucleic acids or proteins that differ in size, charge, or other properties. The separation of the molecules depends on the rate of their movement in a polymeric gel in an electrical field. The distance traveled by a DNA molecule is inverse to its size. In other words, if the DNA molecule is larger in size, then it will travel a shorter distance because it has more resistance due to its size. On the contrary, smaller DNA molecule have less resistance so they travel faster down the gel. The DNA is always placed on the cathode side (negative) because DNA is also negative so when the power source is added the  the DNA will repel away from the cathode and towards the anode(positive) side. Restriction fragments can be used to cut up the DNA molecule into bands.
Purpose:
The purpose of this lab was to use restriction enzymes to sequence DNA. Using single, double, and triple digests, we tried to figure out the number of cut sights present in the DNA sequence for each enzyme as well as the position of those cuts relative to one another.

First our teacher cast an agarose gel with wells included.

Using a pipet we loaded the contents of a reaction tube (DNA with restriction enzymes) into a well in the gel.

We repeated this procedure with each reaction liquid into a different well.

When we finished loading it looked like this.

We put our gel in the electrophoresis chamber, and allowed the DNA to electrophorese until the bromphenol blue band was about 2cm from the end of the gel.

We removed the gel.

And we examined it on a light box. We assigned sizes to the lambda DNA size marker bands, then approximate sizes to the unknown DNA fragments, and determined the total size of digested DNA.

Data and Graphs

This shows the marker's lengths, so that we can
estimate the lengths of the DNA strands for the rest of the lanes.

Discussion: 

 Gel electrophoresis was used to determine the size of the unknown DNA sequence. The size of sequence was determined to be  about 5500 base pairs in length by comparing against a known lambda DNA sequence. When the unknown DNA sample was cut with restriction enzyme PstI, there were two bands at approximately 750 and 4700 base pairs.  The PstI had cut the unknown sequence twice. PstI and SspI there were three bands at approximately  750, 2140, and 2700 base pairs. This shows that the SspI sequence had only cut once. PstI and HpaI there are two bands at approximately 750 and 4700 base pairs.  Since there were only two bands, this shows that adding the HpaI did not cut the sequence. PstI, SspI and HpaI there are three bands at approximately 750, 2858 and 1093  base pairs. This is expected after since PstI cut twice, SspI cut once, and Hpal did not cut at all; giving a total of three cuts and three bands on the gel. Further investigations could look into the discrepancy between the bands in the lane containing PstI and SspI and the lane containing PstI, SspI, and Hpal.

Conclusion: Our results demonstrate how different restriction enzymes cut the plasmid into different sizes. All the different wells should have DNA fragments of the same size because we used the same DNA, but our variables were the restriction enzymes that digested the DNA at different locations. PstI only cut the plasmid into 2. PstI and SspI combined created a plasmid with 3 different sections. PstI and HpaI also created 3 different sections even if they one of them wasn't clearly visible on the gel. The last well with all the enzymes shows a combination of the fragments.