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Bacterial Transformation

Introduction

Biotechnology has to do with the manipulation of organisms to get useful products. One of the basis of biotechnology is genetic transformation. Genetic transformation occurs when DNA is taken in and expressed by a cell from a living organism. Three basic thing are needed to perform genetic transformation: a host, a vector, and a method to select and isolate the transformed organisms. The host is the organism that will take in the DNA. The vector is the means of transporting the DNA into the host. A common method for selecting the transformed organisms is to introduce them to an environment containing what they are resistant to (once they transform) because only the transformed organisms will be able to survive in that environment.

In this experiment, we will perform genetic transformation of the bacteria Escherichia coli (E. coli), therefore it is properly defined as bacterial transformation. E. coli will be our host. It is a good host for transformation because it contains one chromosome and it divides and grows very quickly. The vector in this case will be a plasmid, the simplest bacterial vector. Plasmids are circular DNA that contain important information for the growth of bacteria, in this case genetic information vital for their survival. In this experiment, the plasmid will enter the bacteria cell and insert the gene of interest: resistance to the antibiotic ampicillin. Therefore, the purpose of the experiment is to verify that only transformed bacteria (containing plasmid DNA) will be able to survive and grow in a medium containing the antibiotic ampicillin. To fasten the uptake of plasmid by the bacterial cells, the procedure of heat shock will be performed. The cells will be transferred suddenly from ice to a water bath at a temperature of 42°C. This sudden change in temperature will cause the pores in the cell wall of the bacteria to open, so that the plasmid DNA can enter the cell. Then, the bacteria will be transferred to the ice again so that the pores can be closed and the new DNA synthesized.

Ampicillin is an antibiotic fatal to many bacteria. It affects Gram-positive (largest group of bacteria; blue or violet under a microscope)  and some Gram-negative bacteria (smaller group; red appearance) by penetrating their cell walls and causing cell lysis. As studied in previous sections, cell lysis means that the bacteria cells will burst because the ampicillin concentration will be too high inside the cell. This will induce water uptake by the cell, which will end up dying. E. coli belongs to the Gram-negative bacteria group and it is intolerant to ampicillin. E. coli is a disease causing bacteria. (common meningitis, pneumonia)

Four plates will be used in total for this experiment. The "LB/Amp +", the experimental plate, will contain Luria broth (nutrients), ampicillin, DNA plasmid, E. coli. Hypothetically, because the bacteria cells should be transformed by the vector (plasmid), there should be a bacteria growth in this plate. In the presence of ampicillin and nutrients, the ampicillin resistant E. coli should be able to survive. "LB/Amp -" will be the negative control for transformation. It  contains LB, ampicillin, E. coli, but no plasmid. In the absence of a vector, there should be no growth because the cells are not transformed, and will not tolerate the antibiotic.  "LB +": contains LB and E. coli with plasmid, and therefore ampicillin resistant. In this plate, there should be a big growth because the transformed bacteria are in a nutrient rich medium. Even if some of the cells are not transformed, they would still be viable because there is no ampicillin in the plate. The "LB-" plate contains luria broth and the non-transformed bacteria. There should be regular growth in this plate because normal E. coli cells without ampicillin are able to survive. All these hypothetical assumptions will be tested by the experiment.  
 
Materials
 For this experiment, the following materials were used:
- Two eppendorf tubes
- Transfer pipettes
-  A container with ice
-  Ice-cold calcium chloride
- Escherichia coli (E. coli) sample
- Plasmid DNA sample
- Plastic loop
- 4 starter plates (Petri dishes)
- A microcentrifuge
- Luria broth (LB)
- Ethanol
- A cell spreader
- A Bunsen burner
- Parafilm

Method
1. Two eppendorf tubes were marked "+ DNA" and "- DNA", respectively.
2. 250 uL of cold calcium chloride was added to each tube using a transfer pipet. Then the tubes were placed on ice for about 2 minutes.
3. A small cell mass of E. coli colonies was transferred to the "+ DNA" tube using a plastic loop. Then the tube was tapped until the cell mass disintegrated. After that, the cells were suspended by pipetting the solution in and out with the transfer pipette. Then the tube was returned to ice.
4.  The same procedure (as in step 3) was performed for the "- DNA" plate. The tube was returned to ice with the "+ DNA" plate.
5. 10 uL of plasmid was added to the "+ DNA" tube. The contents were mixed by tapping the tube gently and  returned to ice. Then, both tubes were incubated for 30 minutes.
6. During the incubation time, the plates were labeled with the group date, section and date. The plates were labeled "LB/Amp +", "LB/Amp -" ( Luria broth and ampicillin in both), "LB +" (one half of the class) and "LB -" (the rest of the class. Assigned to our group)
7. After the incubation time was up, a heat shock was performed to the E. coli cells. The tubes were taken out of the ice and placed in a 42°C water bath for about 90 seconds, where they were agitated every 10 seconds. Then, the tubes were placed in the ice container for 2 more minutes.
8. The test tubes were centrifuged for 1 ½ minutes at 6000 rpm. The liquid in the tubes, the supernatant, was poured out without moving the cell pellet at the bottom.
9. Then 250 uL of Luria broth (LB) was added to each tube. The cells were re-suspended as in step 3 and 4. After that, the tubes were incubated at room temperature for 30 more minutes.
10. Then, 100 uL from each tube was placed on the plates. Contents from the "+ DNA" tubes were placed in the "LB/Amp +" plate,  and cells from the "- DNA" one placed in the "LB/Amp -" and "LB - " plates. (Our group was not assigned with the "LB+", but some people were)
11. A metal spreader was dipped in ethanol, and passed through a flame. It was let cool down for 2 minutes and then used to spread the cell solution evenly over the surface of the starter plate. Then the plate was covered with its lid.
12. The same procedure as in step 11 was performed for the two remaining plates, which were let cool for about five minutes before parafilm was wrapped around them. The group # and section were written on the top of the plates. Then, the plates were wrapped together with parafilm, and placed in the incubator.

Results

The plates were checked after a week. During this period of time, they had been incubated and refrigerated. The following drawings indicate the results regarding amount of growth, if any, for each plate "LB/Amp +"  Experimental plate for transformation.  { luria broth, ampicillin, DNA plasmid, E. coli }

In the "LB/Amp +" plate, bacterial growth was observed. The transformed E. coli colony (white/gray color) occupied about ¼ of the total surface of the starter plate. The ampicillin resistant bacteria are able to grow in an environment where ampicillin is present. However, the amount of growth was less than in the "LB +" plate. "LB/Amp - "Negative control for transformation. {luria broth, ampicillin, E. coli, no plasmid}

In this plate, no growth of bacteria was observed. There was no cell mass in the plate because the bacteria got killed by the antibiotic. It was found out that the non-transformed bacteria cannot survive in an ampicillin containing environment. "LB + " Positive control for viability of cells. {luria broth, E. coli, DNA plasmid}

The greatest growth in cell mass was observed in this plate. Our group was not assigned to do this plate so the results were gathered from the groups that did perform it. A white/gray cell mass occupied abundant space in the surface of the plate. It was observed that the transformed bacteria, and therefore ampicillin resistant, are viable and can grow in a normal nutrient containing environment.

"LB -"  Negative control for cell viability. {luria broth, E. coli cells without DNA plasmid}

There was sufficient growth in this plate, but less than in the "LB +" one. It was found out that without the plasmid DNA, the regular E. coli cells (nonresistant to ampicillin) are viable, and are able to grow in a normal environment with sufficient nutrients.

Discussion

In this experiment bacterial transformation was observed using E. coli as a host and plasmid as a vector. It was found out that the transformed bacteria are able to survive in an ampicillin rich medium, and that the non-transformed ones are not able to grow.

There was growth in the "LB/Amp +" plate because the plasmid DNA was added to the E. coli cells, making them ampicillin resistant. The way it works is that the vector carries the ampicillin resistance gene, and it inserts it into the bacterial DNA. The newly "transformed" cells are now resistant to the antibiotic, and can therefore survive in the plate containing ampicillin. This is what I predicted in my introduction, where I stated that the transformed cells should have no problem surviving in the antibiotic containing plate. On the other hand, there was no growth in the "LB/Amp -" plate because the E. coli cells did not contain plasmid DNA so they did not grow resistant to ampicillin. When they were placed in the plate containing ampicillin, they could not survive because that antibiotic is lethal to the bacteria. This coincides with my prediction about the non-transformed bacteria, stating that the E. coli cells without plasmid would not grow resistance to the antibiotic and would get killed when placed in the plate.

The "LB +" and "LB -" plates were the positive and negative control for the viability of the cells, respectively. The "LB +" plate is the positive control because it shows how the ampicillin resistant cells function under normal circumstances, where there is nothing that can get them killed. There was growth in this plate because the cells are all viable, and can easily grow in a nutrient containing medium. In the introduction, I predicted that there would be a very big increase in cell mass in this plate. The results show that I was correct on that assumption and that E. coli grew the most in this plate, even though I cannot be one-hundred percent sure of why it turned out that way. One possibility is that the transformed bacteria did not have anything against them, in terms of substances that could get them killed. Because they are antibiotic resistant, they can grow even if some antibiotic were there by mistake. Another reason could be that the plasmid DNA, being part of the bacterial DNA, is more efficient when it comes to cell division, and that it somehow shortens the span of the mitotic cycles so that more cell cycles are performed in the same amount of time.

Lastly, there was also growth observed in the "LB -" plate. It was the negative control because the E. coli cells have not been transformed. The cells did not contain the plasmid DNA, and were not ampicillin resistant. However, this did not affect the growth of the cells because there was no antibiotic in the plate, only luria broth. In a nutrient rich environment, the regular (non-transformed) E. coli cells are viable and can survive. This is consistent with my hypothesis in the introduction, where I stated that E. coli cells in this plate should have no problem growing. There was less amount of cell mass in this plate than in the "LB +" plate. The reason could be that any advantage given by ampicillin resistance in a normal environment to the "LB +" cells are not given to the cells in this plate because they were not transformed.

Overall, the main purpose of the experiment was achieved. It was observed that both the transformed and non-transformed E. coli cells are able to grown in a nutrient rich environment lacking ampicillin, but that only the transformed cells can survive in an ampicillin containing medium. The key to this resistance lays in the plasmid DNA because it contains the ampicillin resistance gene. When the plasmid DNA combines with the bacterial DNA, the bacterial DNA gets transformed into an ampicillin resistant bacterial DNA. The E. coli cells that lacked the plasmid cannot survive in the ampicillin containing plate because they were not transformed, and their DNA does not contain the ampicillin resistance gene.

A source of error for this experiment could be that when the cell spreader is dipped in ethanol and passed through the flame, it is supposed to be let cool down for at least two minutes. If it gets in contact with the bacterial cells in the plate without being cooled, then it would kill many of the cells because the bacteria are affected by high temperature. This would cause less growth in the plate.

A second source of error is the heat shock itself because this procedure is not 100 % efficient. The uptake of plasmid DNA by the bacterial cell depends on the time they are let in the water bath. If they are removed too fast (before the 90 seconds), then the rate of uptake would be smaller. Also, it is not 100 % certain that the plasmid DNA would enter every single cell wall, and so not every bacterial cell will be transformed. Any cell that is not transformed will not survive in the ampicillin containing plate, and this would cause less growth in that particular plate.

References
Raven, Peter H., and Cleveland Hickman. Biology: A customized version. DNA: The genetic material. Boston: McGraw-Hill, 2004.p282

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