1.0 Introduction and Objectives
The ability of bacteria to incorporate DNA from external sources is the primary reason for their survival and proliferation. Bacteria can take DNA from their surroundings or from other bacterial cells by cell wall-transfer. While an interesting phenomenon to examine for scientists, practically it is of great concern for the human race and a source of constant challenge for the Pharmaceutical Industry. The ability of bacteria to modify their genetic information has given rise to problems such as antibiotic resistance wherein bacteria become resistant to medications that were once effective in eliminating them. In this experiment, we examine the development of antibiotic resistance in bacteria. Circular DNA called plasmids are introduced in bacteria whose cells have been modified to promote uptake of plasmid DNA. This plasmid DNA will give rise to antibiotic resistance in the bacteria, which can be observed by allowing the bacteria to proliferate in an environment containing the antibiotic.
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Modification of genetic information in bacteria may be a source of concern, but that ability in the hands of humans has always been coveted. Genetic engineering is an increasingly popular research area given the breakthroughs made in recent years and the potential for commercial application. Various applications require large quantities of specific DNA sequences and this is where the bacterial ability to uptake DNA and reproduce it is beneficial. Introducing plasmids containing desired sequences into bacteria, allowing bacteria to reproduce and then isolating the required DNA is a common method used to obtain large quantities of particular DNA sequences. This aspect is also explored in this experiment.
The objectives of this experiment are to:
a) Observe and examine the phenomenon of DNA Transformation.
b) Observe the development of antibiotic resistance in bacteria through the process of gene transformation.
c) Inculcate proper Sterile Technique for laboratory procedures involving bacterial strains.
This section explores the underlying concept behind the experiment.
Genetic Transformation is a process of horizontal gene transfer whereby DNA from the environment is taken up by a host cell. In this experiment bacterial cells are transformed. Escherichia Coli bacteria, which are generally non pathogenic are used in this experiment. The plasmids which constitute the external DNA contain a gene that makes the cell ampicillin resistant. Ampicillin is a bacteriostatic and will normally prevent the reproduction of E. Coli bacteria. This provides us with an easy way to test if gene transformation has occurred and to what extent by means of calculating the transformation efficiency.
The introduction of genetic material within the bacterial cell is done by the process of electroporation. Electroporation involves applying an electrical voltage across the bacterial cells containing the plasmids. The ionic concentration of the DNA is kept low to prevent arcing. When the voltage is applied, holes open up in the walls of the bacteria. The plasmids can then enter the bacterial cells through these holes. Application of the voltage is done for a very short period of time. As soon as electric current stops flowing, the holes in the cell wall begin to close. A nutrient rich medium is then added to the bacterial cells, some of which will have transformed, to aid cell recovery.
Incubation is then carried out, after which the cell suspension is diluted further and applied to agar plates containing the antibiotic. The cells are left to incubate for up to 24 hours and then the number of colonies determined. Calculating the transformation efficiency gives us a method to determine the extent to which the transformation occurred.
3.0 Methods and Materials
The equipment and materials required for this experiment are outlined in this section.
* A shaking incubator operating at 37ÌŠC
* A non-shaking incubator
* An electroporator
* Cells treated for competency
* 2 agar plates with ampicillin with a concentration of 100 µg/ml
* pUC-19 plasmids
* 0.1 cm cuvettes
* Ice in an ice-box
* Deionised ultrapure water
* S.O.C. medium at room temperature
* 2 tubes with snap caps with a volume of 15 ml
3.2 Sterile Technique
Sterile Technique is a must when handling pathogenic strains of bacteria. In this experiment, nonpathogenic bacterial strains are employed. However, using sterile technique is still good experimental procedure and promotes safety.
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Using sterile technique prevents errors in experimental results by preventing contamination from the surroundings. It also prevents contamination of the surrounding environment by the bacterial strain. Steps employed to prevent contamination included:
· Carrying out the experiment in an uncluttered area.
· Utilizing a fume hood to perform all procedures involving the bacteria.
· Washing hands both before as well as after the experiment
* Disposing off all bacterial waste in the appropriate container for bio-hazardous materials.
3.3.1 Preparation for Electroporation
1. The 0.1 cm cuvettes were cooled on ice.
2. The electroporator was prepared based on prescribed settings.
3. In order to bring the S.O.C. medium to room temperature, it was removed from the ice box.
4. The cells and plasmids were allowed to thaw in the ice-box.
5. Plates were heated at 37ÌŠC to prepare for the incubation process.
1. I µl of pUC19 control DNA and 1 µl of ultrapure water were added to 2 separate microcentrifuge tubes with the aid of a pipette. The tube was then placed in the ice-box.
2. 25 µl of competent cells were added to each of the microcentrifuge tubes. The contents of the tubes were gently mixed. Care was taken to avoid usage of the pipette for mixing. The tubes were then returned to the ice-box for 1 minute.
3. The contents of each microcentrifuge tube were transferred to a cuvette using a pipette. It was ensured that the cells made contact with the cuvette walls and that no air-bubbles were present. This step was done rapidly to prevent heating up of the cells.
4. The cuvettes were then electroporated.
5. 250 µl of S.O.C. medium was added to the cells immediately after electroporation.
6. Each of the two suspensions was transferred to a 15 ml tube. The shaking incubator was then set to 225rpm and used to incubate the cells for an hour to allow expression of the acquired antibiotic resistance.
7. 10 µl of the transformed sample was then added to 90 µl of S.O.C. medium.
8. The plates containing the ampicillin were then used. 20 µl of each of the two diluted samples from step 7 was added to a plate. Even spreading of the sample on the agar medium was ensured.
9. Using the non-shaking incubator, the plates were incubated at 37 ÌŠC for a day and the results recorded.
4.0 Results and Discussion
4.1 Results - Answers to Questions
(1) Schematic of observations of the agar plates:
Figure 1: Results as Indicated by the Agar Plates
(2) Count the colonies and calculate the transformation efficiency.
Number of colonies observed = 13
Figure 2: Calculation of Transformation Efficiency
Using the formula shown in figure 2,
Transformation efficiency = 1.78 - 1010 transformants/µg plasmid DNA
4.2 Discussion - Answers to Questions
(1) Define the vocabulary used in this experiment: transformation, electroporation, host, plasmid, and competent.
Transformation is a process of horizontal gene transfer whereby DNA present in the environment of a cell is taken up by the cell. In this experiment the transformation involves the uptake of a plasmid containing a marker that results in ampicillin resistance by E. Coli bacteria through electroporation.
Electroporation involves subjecting cells to an electric voltage to create holes in the cell wall. External material can then enter the cell through these holes. Natural processes then cause the hole to close and return the cell to its original state.
An organism that harbours a parasite is called a host.
A plasmid is circular extra-chromosomal DNA.
A competent cell is one which can internalise DNA present in its external environment. Competence can either be natural or artificial.
(2) State why E. coli is used in many genetic engineering experiments.
The popularity of Escherichia Coli for genetic experiments is due to various reasons. Firstly, most E. Coli strains are non-pathogenic and pose no harm to humans. Safety is a significant factor in the laboratory and E. Coli use is generally safe. Secondly, E. Coli grow easily and can be duplicated through metagenics. Thirdly, their genetic make-up is relatively simple and can be manipulated with ease. Fourthly they have been extensively studied and a lot is known about them. This makes it easier for researchers and they therefore prefer to use E. Coli for genetic engineering experiments.
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(3) Explain why competent cells, ampicillin, and S.O.C. medium were used for the transformation.
Competent cells are necessary as transformation involves taking external genetic material into the cell. If cells are not competent this cannot happen and the experiment cannot be carried out successfully.
Ampicillin is an antibiotic. Specifically, it is a bacteriostatic for E. Coli. It helps distinguish between bacteria that have taken up the plasmid and those that have not. This is because the plasmid contains a marker that causes ampicillin resistance. E. Coli cells do not naturally contain the genetic sequence that causes ampicillin resistance. Thus, ampicillin selection is possible to distinguish between transformed cells and untransformed cells.
S.O.C. medium contains the nutrients required to help cells stabilise after electroporation. Electroporation introduces holes into the cell wall of the cell and therefore causes destabilisation of the cell. S.O.C medium contains yeast extract and other nutrient sources that help the cell recover. Once the cell has recovered and if the plasmid has entered the cell during electroporation, the cell will multiply and give rise to a colony during the incubation period.
(4) Explain the purpose of the controls in this experiment.
The control in this experiment constitutes bacteria without the plasmid that inculcates antibiotic resistance. Without this extra piece of genetic information to enable the bacteria to mount defences against the attack of the antibiotic, ampicillin is this case, the bacterial cells will be unable to multiply in a medium that contains the antibiotic. The cells that were treated such that they could incorporate the plasmid DNA into their genetic make-up will be able to multiply in a medium where ampicillin is present as long as there are enough nutrients available for growth. Thus, the control helps us show that the DNA plasmid was indeed taken up and incorporated into their genetic make-up by the bacteria. The only way for E. coli to have survived with ampicillin present is if they had taken up the plasmid and transmitted it to all generations when they reproduced after uptake of the plasmid. Hence, the control serves to confirm uptake of the plasmid as well as its transmission to following generations by comparing it to cells in the control that did not have the extra DNA.
(5) Explain how the colony growth relates to gene transformation.
A colony of bacteria stems from the binary fission of one single bacterial cell. When bacteria reproduce vertical genetic transfer occurs whereby the offspring has the exact copy of the genetic material of the parent.
In this experiment, bacteria are introduced into a medium containing the antibiotic ampicillin. E. Coli bacteria with their original genetic make-up will be unable to reproduce due to the presence of the antibiotic as they do not have the means necessary to resist antibiotic attack. This is what is expected in the control sample as ampicillin is a bacteriostatic.. The positive sample on the other hand has bacteria which have undergone horizontal gene transfer by transformation. The plasmid DNA that was used for the transformation process contains genetic code that results in E.Coli developing ampicillin resistance. Thus, bacteria that can incorporate this plasmid and pass it on to their offspring by vertical gene transfer can grow in the environment. This is how colony growth relates to gene transformation.
(6) Describe how ionic strength of DNA solution affects electroporation.
The ionic strength of DNA solution comes into play due to the electroporation stage where holes are created in the bacterial cell wall to allow uptake of the plasmid by transmission of an electric voltage. For this step, the ionic strength of the solution must be low. If the ionic strength is high, arcing will occur. Arcing is visible during the experiment by sparks and a sound like a micro-scale thunderclap. It can cause cell death as well as equipment damage. Thus, for the experiment to be carried out successfully and to safeguard the apparatus, the DNA solution must be of low ionic strength.
(7) If your transformation efficiency is lower than 1 - 109 cfu/μg, conjecture and explain potential reasons for the low efficiency.
The transformation efficiency is greater than the benchmark stated above. This corresponds to good transformation efficiency and indicates a successful transformation process. However, the close clustering of the colonies makes it possible that some of the colonies are satellite colonies rather than transformed colonies. The experiment could be repeated with a higher concentration of ampicillin to obtain more reliable results.
(8) Discuss current and potential applications of gene transformation techniques in biotechnology.
Gene transformation techniques play a crucial role in biotechnology. This is because gene transformation provides a method to produce copies of desired DNA sequences. This is especially useful in the pharmaceutical industry to develop medications that are target specific. Also, this could potentially lead the way to genetic engineering, where defects to the genetic code could be repaired and desired traits inserted through addition of the corresponding DNA sequences. Gene replacement therapy could prove to be the cure for nearly all diseases that take human lives contemporarily. In the future gene transformation could be used to engineer human beings and other animals and plants according to desired specifications.
Genetic transformation is also used in the development of pest-resistant crops, which could potentially increase the productivity of the land. This could be key to feed the ever-growing population as the quantity of agricultural land decreases.
Understanding the evolution of drug resistance could help us devise ways of preventing drug resistance as well as developing drugs that can overcome resistance. In this arena gene transformation plays an important role horizontal genetic transfer is a natural process in bacteria.
4.3 Sources of Error and Suggestions for Improvement
There are a few sources of error that could result in incorrect conclusion being drawn from experimental results.
(i) The number of colonies seen need not correspond to the bacteria that transformed. This could be due to the growth of satellite colonies. Large bacterial colonies will secrete beta lactamase, which is what causes ampicillin resistance. Thus, the area around the colony will contain this secretion and be ampicillin-free. A satellite colony could grow in this area from untransformed cells.
To avoid this problem, the incubation period should strictly be restricted to 24 hours. Satellite colonies emerge after a delay. By ensuring that results arr recorded promptly, the interference in results brought about by satellite colonies can be minimised. Another method is to use a higher concentration of ampicillin. More time will be required to create a antibiotic-free zone around a colony if the concentration of antibiotic is high.
(ii) Identifying the number of colonies can be difficult, especially if the size of the colony is miniscule. This could result in an incorrect calculation of transformation efficiency.
In order to increase accuracy of results, a different selection marker can be used. Some selection markers have properties that can be distinguished by shining UV light and other such techniques which result in a high contrast. Using these markers may result in higher reliability of results.
(iii) Distinguishing between colonies can be difficult if they grow close to one another and appear to be one large colony. Also, closer colonies would also result in a higher chance of there being satellite colonies.
To minimise this problem, crowding on the plate must be minimised. For that, a higher concentration of ampicillin could be used, carbenicillin selection could be used instead of ampicillin selection (although expensive) or the nutrient dilution could be adjusted such that it discourages very rapid proliferation.
The objectives of this experiment were to explore the phenomenon of gene transformation and the development of antibiotic resistance in bacteria as well as to inculcate the practice of sterile technique for handling bacteria. Gene transformation was observed with the development of ampicillin resistance in transformed Escherichia Coli bacteria. The bacteria not exposed to the plasmids containing the genes for antibiotic resistance did not grow in an environment containing the antibiotic while the transformed bacteria formed colonies in the same environment. A calculation of transformation efficiency returned a value of 1.78 - 1010 transformants/µg of plasmid DNA, which is greater than the threshold of 109, indicative of a successful experiment. However, the possibility of some of the 13 colonies of bacteria being satellite colonies as opposed to transformed colonies reduces the reliability of the results. Methods to increase reliability of results were therefore suggested.
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2. Metzenberg, Stan. (2002). Bacterial Plasmids. California State University Northridge Department of Biology. Retrieved 4th April, 2010 from http://escience.ws/b572/L2/L2.htm