Sunday, 15 January 2012


pGLO Bacterial Transformation Using Calcium Chloride Transformation Solution and Heat Shock


IDC-4U5

co-authors : Katelyn Dixon
Eric Horton
Sharmarke Mohamed
Jordan Khan


Abstract 
Genetic transformation occurs when an organism is modified by the introduction of new genetic information which is incorporated into the organism’s genome. Bacterial transformation is the easiest type of genetic transformation to create in a lab due to the single celled nature of bacteria. In this lab the engineered pGLO plasmid is incorporated into E. Coli bacteria, and adds the genes which code for the proteins GFP and beta lactamase to the modified bacteria’s genome. To see the effects of this plasmid on the cells, bacteria treated with the plasmid were grown on two separate agarose plates containing LB nutrient broth and ampicillin, and another containing LB nutrient broth, ampicillin and arabinose. To contrast these plates two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that didn’t contain the plasmid. To see the effects of the calcium chloride solution used in the procedure another experiment was run under the same parameters, except the calcium chloride solution was replaced with distilled water. The results showed that when calcium chloride was used the plasmid was successfully incorporated into the E. Coli’s genome, since the cells treated with the plasmid fluoresced green under ultraviolet light and were resistant to ampicillin, an affect of beta lactamase. The results in the second test showed that the calcium chloride solution is essential to efficient plasmid uptake, but to what degree was not determined. Transformation efficiency was used in this lab to quantify the uptake of the plasmid DNA. In the tests where calcium chloride was used the transformation efficiency was much higher than those where distilled water was used. In a further analysis of the experiment sources of error, future prospects, and the ethical implications of bacterial transformation are discussed.  

Introduction 
Bacterial transformation occurs if a bacterium uptakes a piece of DNA and integrates it into its genome. Bacterial transformation is done in two different ways in the lab: electroporation and through calcium chloride/heat-shock. Electroporation is when electrical shock is used to make the cell membrane permeable to DNA. Calcium chloride/heat-shock is when the plasmids are incorporated into the chemically-competent cells made permeable by the heat shock and calcium chloride solution.  In 1928, Frederick Griffith, a physician from London, was the first person to experiment with bacterial transformation.  He permanently transformed a safe, nonpathogenic bacterial strain of pneumococcus into a deadly pathogenic strain.[6] 
Escherichia coli or E. coli is a gram negative, bacillus, bacteria. E. coli makes a great bacterial candidate for the bacterial transformation because it is made up of one cell, it reproduces every twenty minutes, it does not harm people, and it cannot survive outside of the lab. Ampicillin is an antibiotic that has a beta-lactam structure. It has the ability to kill E. coli cells. [7] 
Plasmids are small circular autonomously replicating pieces of DNA, found inside prokaryotic bacterial cells. They replicate their own DNA by borrowing the cells’ polymerase. Due to their size, plasmid DNA is easy to extract and purify from bacterial cells. Plasmids may express antibiotic resistant genes or be modified to express proteins of interest, and  are useful for cloning foreign genes. [8] 
The pGLO plasmid contains the gene for green florescent protein (GFP) and a gene for ampicillin resistances called beta-lactamase . The pGLO plasmid also contains a special gene regulation system,  or operon, that can be used to control expression of the florescent protein in the transformed cells called araC regulator protein. [6][7] 
Green fluorescent protein, or GFP, is found naturally in the luminescent jellyfish Aequorea Victoria. GFP glows bright green when exposed to ultraviolet light due to resonation which the light causes. GFP is mainly used in biotechnology as a biological marker or indicator. It has also been used to produce luminescent plants and animals.[4]                      GFP production is monitored by a modified arabinose operon located on the pGLO plasmid. In the operon, the protein araC blocks RNA polymerase from binding to the Pbad promoter. When arabinose is present it changes the shape of the araC protein causing it to promote, instead of prevent, RNA polymerase binding. Once RNA polymerase has attached to the promoter, transcription of the GFP gene begins and continues until arabinose runs out.[9][3]         
 Ampicillin is an antibiotic that falls into the penicillin group of drugs. Ampicillin is often used to fight bacteria in the human body, specifically various types of infections. Such infections may include  those  caused by bacteria, like ear infections, bladder infections, pneumonia, gonorrhea, and E. coli or salmonella infection. Ampicillin’s ability to exterminate bacteria makes it ideal for this lab [10].                      
During the course of this experiment, our objective was to manipulate a variable present within the lab and determine what affect this change would have on the results of the lab. Our particular objective was to determine whether or not the calcium chloride transformation solution was a critical ingredient in influencing the uptake and expression of the pGLO gene. In order to accurately test this variable, we performed the complete pGLO lab as instructed in the pGLO manual as a control, and then proceeded to manipulate the lab through duplicating the lab setup, however using an altered transformation solution. The calcium chloride was replaced with distilled water for the second set of plates, in order to compare the expression of the pGLO gene in each case. In the end there was a total of 8 petri dishes. The first four were prepared ahead of time, and filled with a solution containing nutrient agar solution, as well as a combination of various solutions. These solutions included LB broth, ampicillin and arabinose. Two of the plates, (LB/amp) and (LB/amp/ara) we covered in a solution consisting of transformation solution, LB broth and pGLO plasmid DNA. The other two, (LB/amp) and (LB) were covered in a solution consisting of only the transformation solution and LB broth. These four plates acted as our control. We anticipated that the LB/amp/ara plate containing the pGLO plasmid DNA would become fluorescent. The other four plates were the exact same, except none of them were covered with a solution that contained the calcium chloride; instead distilled water was combined with the LB broth and pGLO plasmid DNA. It was anticipated that in the plates not containing the transformation solution, there would be no expression of the pGLO gene. This result is expected because in theory, the transformation solution is an essential step for giving the bacteria the ability to take up the pGlo plasmid, since the solution makes the bacterial cell walls more permeable. 

Methodology


Results

The lab results can be seen in the appendix section. As seen in figure 1, the control test, the LB plate containing the -pGLO sample, bacteria grew evenly on the plate in the areas where it was spread with the transfer loop. In the LB/amp plate treated with the -pGLO sample absolutely no bacterial growth was observed. In the LB/amp plate containing the +pGLO sample several large, yellow colonies appeared, some grouped in clusters and others spread over the plate. In the LB/amp/ara plate with the +pGLO sample many yellow, differently sized colonies grew, which fluoresced green under ultraviolet light. 
Figure 2  contains the results for the variable test, where the transformation solution was replaced with distilled water. In the LB plate containing the -pGLO sample, bacteria grew evenly on the plate in the areas where it was spread with the transfer loop. In the LB/amp plate treated with the -pGLO sample absolutely no bacterial growth was observed. In the LB/amp plate containing the +pGLO sample a few small yellow colonies appeared, spread randomly across the plate. In the LB/amp/ara plate containing the +pGLO sample no bacterial growth was observed. 
When the transformation solution was used, there were 492 transformants per micro gram, as seen in  figure 2  of the appendix section. However, when the transformation solution was replaced with distilled water, there were 0 transformants per micro gram, as seen in figure 1 of the appendix section.  
Figure 3 of the appendix section shows the calculations for the transformation efficiency on the LB/amp plate where no transformation solution was used. There were 67 transformants per micro gram on the LB/amp plate.

Discussion 

                In the control lab a different outcomes was observed in each of the four plates. In the LB/amp/arabinose agarose plate containing the +pGLO sample, fluorescent green colonies developed. This is because the gene which codes for the fluorescent protein, GFP, is located near the beta lactamase gene on the pGLO plasmid, which protects bacteria from the antibiotic ampicillin. When the cell produced beta lactamase to deactivate ampicillin, the GFP gene was also transcribed, producing the fluorescent protein observed. In the LB/amp plate containing the +pGLO sample white, non florescent cells were observed. While these genes contained the pGLO plasmid and the GFP gene they could not express the GFP gene because they were not grown in the presence of arabinose. While the presence of ampicillin causes the cell to transcribe the beta lactamase and GFP genes, arabinose is needed to activate the GFP operon. Therefore without arabinose in the agarose gel, GFP cannot be transcribed and the cells will not fluoresce. In the LB/amp agarose plate treated with the -pGLO sample, no cells grew. This is because without the pGLO plasmid and the beta lactamase gene the cells cannot deactivate the ampicillin in the gel. Therefore all the cells were wiped out. In the LB plate containing the -pGLO sample small colonies were seen spread over the entire plate. Because there was no ampicillin to kill the cells, all the cells survived and the entire plate was covered, in contrast to the other plates where individual colonies represented the cells which had taken up the plasmid. 
 In the variable lab, where the calcium chloride solution was replaced with distilled water, results for the -pGLO samples were the same. This is because the calcium chloride solution affects plasmid uptake and no plasmid was introduced to these samples. On the LB/amp/arabinose plate containing the +pGLO sample no bacterial growth was seen. This is because without the calcium chloride solution to make the cell walls of the bacteria more permeable the plasmid was not taken up by the cells. Without the beta lactamase gene to deactivate the ampicillin all the cells were killed and no colonies developed. In the LB/amp plate containing the +pGLO sample a small amount bacterial growth was seen. This means that some of the bacteria was taken up by the cell, since the beta lactamase gene prevented the ampicillin in the plate from killing all the bacteria cells. It is unknown why there was no plasmid uptake in the LB/amp/ara plate but some in the LB/amp plate. It may be due to an inconsistency in the procedure, or a random occurrence. Due to the varying results in this test, the affect of replacing the calcium chloride solution with water cannot be definitively stated, but it is known that it reduces plasmid uptake. 
The transformation efficiency of our control test was 492 transformants per microgram. This is much lower than the average of between 800 and 700 transformants per microgram. This lower number could be a result of sources of error that may be present within the methodology of this lab or potential human error. Our variable test had an even lower transformation efficiency of 0 transformants per microgram. This could be attributed to the lack of transformation solution, but the tests should be repeated to reach a definitive result. 
                Due to the multiple solutions and bacterial plates used in this lab there it is likely that some cross contamination occurred. Though many precautions were taken, such as using disposable pipettes and sterile loops, there is always a chance that these tools could be contaminated before use, or that a new substance, such as bacteria, was introduced from the environment. While this could be improved by using a culture hood or wearing gloves, cross contamination, especially from the environment, can never fully be prevented. 
                In this lab transformation efficiency was used to measure how successfully the plasmid was incorporated into the bacterial cells. While calculating transformation efficiency it was found that it depended highly on the amount of bacteria taken from the starter colony. Because of this inaccuracy the success of the experiment cannot be accurately quantitatively described. Even if only one colony is taken, it is unlikely that any two colonies are exactly the same size. 
                The transfer pipettes used during much of the procedure weren’t very accurate, since any variation of the pressure applied to the pipette would change the volume. This inaccuracy could be eliminated by using micropipettes, which are accurate to the microliter. 
                In the variable lab, the lack of calcium chloride solution had different effects on both +pGLO plates. On the LB/amp/ara plate no plasmid was incorporated into the cells, but on the LB/amp plate the plasmid was at least partially taken up by the cells. The reason for this inconsistency is unknown but is probably due to some error made in the methodology. This might have been because of cross contamination or incorrectly timing the temperature shocks used. To determine the cause of this anomaly the variable lab could be repeated to see if the results were similar. 
                This lab has many future prospects. Genetic modification does not have to be limited to florescence. Organisms can be modified to have all sorts of interesting and unique traits. For example, plants can be given plasmids so that they gain certain traits, such as resistance to disease or extreme weather. This can lead to better crop yield and shelf life. A lab extension of the pGLO lab may involve modifying more complex organisms than bacteria. For example, one could attempt to influence the expression of the GFP gene in fish, or another multi cellular organism. Another potential lab extension might include the isolation and purification of GFP. This could be done using column chromatography. It may also be worthwhile to repeat the experiment without the transformation solution to ensure the results are the same. This would further explain the effect of the calcium chloride solution. 
There are many ethical dilemmas associated with this lab because of the nature of this experiment. During this experiment, living organisms are being altered. Many would argue that in doing so the experimenters are “playing god”. This may seem like an absurd thing to question, but this is a living creature that simply just has no way of expressing itself to us. Do single celled organisms have fewer rights than us? Why should we be able to grow and kill these organisms at our own discretion? Who decides whether or not it is just to alter or work with certain organisms? The opinions on such a subject are very diverse, leaving us with no definitive answer. Many ethical dilemmas like these become evident when working with and altering living organisms for the sake of scientific inquiry; however there are many positive benefits to genetically engineering bacteria. For example, scientist have been able to genetically engineer forms of E. coli so they secrete proteins that have been found to block HIV from infecting cells of other living organisms [2 ]. Researchers at Tel Aviv University have even been able to manipulate bacteria so they light up when they come in contact with certain pollutants in water [1]. Bacteria have been genetically modified for many different reasons; many of them have had an incredibly positive impact on the world around us. As long as the benefits of bacterial transformation continue to outweigh the risks, their use will remain extremely important to humanity and beyond. 


 References


[1] Blajchman, A. (2009, March 20). Genetically Engineered Bacteria to Measure Water Quality. Clean Tech News & Views. Retrieved January 15, 2012, from http://cleantechnica.com/2009/03/20/genetically-engineered-bacteria-to-measure-water-quality/

[2] Bacteria modified to combat HIV. (2005, November 13). BBC News. Retrieved January 15, 2012, from  http://news.bbc.co.uk/2/hi/health/4692905.stm
[3] General Applications of GFP. (n.d.). Green Fluorescent Protein. Retrieved January 15, 2012, from userpages.umbc.edu/~jili/ench772/application.html 
[4] Goodsell, D. (n.d.). Green Fluorescent Protein (GFP). Protein Data Bank. Retrieved January 15, 2012, from www.rcsb.org/pdb/101/motm.do?momID=42
[5] Gregory, M. J. (n.d.). Bacterial Transformation Lab. The Biology Web. Retrieved January 15, 2012, from faculty.clintoncc.suny.edu/faculty/michael.gregory/default.htm
[6] Hanahan, D. (1983). n.a.. Studies on transformation of Escherichia coli with plasmids (pp. 166, 557). n.a.: n.p..
[7] Hanahan, Douglas, Techniques for transformation of E. coli. In DNA Cloning: A     Practical Approach
(Ed. D. M. Glover), vol. 1. IRL Press, Oxford (1987).
[8] Mulligan, M. E. (n.d.). The Arabinose Operon. Memorial University. Retrieved January 15, 2012, from www.mun.ca/biochem/courses/3107/Topics/Ara_operon.html
[9] Schleif, Robert, Two positively regulated systems, ara and mal, In Escherichia coli and Salmonella,
Cellular and Molecular Biology, Neidhardt. ASM Press, Washington, D.C. (1996).
[10] ampicillin medical facts. (n.d.). Drugs.com | Prescription Drug Information, Interactions & Side Effects. Retrieved January 15, 2012, from http://www.drugs.com/mtm/ampicillin.html


Appendix

Table 1
Table 2
Figure 1
Figure 2





Figure 3

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