Identifying Kanamycin Resistant Plasmids Using Restriction Enzymes and Gel Electrophoresis

By: Wayo, D.

Abstract

Since the original discovery of Penicillin, by Alexander Flemming in 1928, antibiotics have helped millions of people fight potentially fatal diseases.Ê Just as companies began mass producing Penicillin, strains of bacteria began to surface that could survive even when introduced to antibiotics. The most common way bacteria is able to resist antibiotics is through a ring of DNA called a plasmid.Ê Plasmids can be easily transferred between two touching bacteria through a process called conjugation (Lewis R. 1995).Ê In this research project, Kanamycin resistant bacteria were found on a sink in 261 East Holmes Hall, which was then cultured to determine the type of plasmid it contained.Ê It is my prediction that the antibiotic resistant bacterium was found on the sink due to the vast amount of antibacterial soaps placed around campus.Ê The bacteria were cultured in agar plates containing Luria-Burtani broth.Ê On the plates was also the Kanamycin, to make sure the bacteria were resistant to the antibiotic.Ê The colonies were then taken off of the plate and cultured to attain a large amount of bacteria.Ê These bacteria were then lysed, leaving only the plasmid DNA.Ê Restriction enzymes were run on the plasmid, cutting the DNA sequences into smaller segments, therefore allowing us to then run them through a Gel Electrophoresis.Ê The banding the plasmids made allowed us to determine the type of plasmid the resistant bacteria contained.Ê The number of base pairs the plasmid had was 3138 and could be the plasmid pUMVC2, pACYC177, pGPS3, pBR322, or pUC18/19.

Discussion

ÊÊÊÊÊÊÊÊÊ Since the discovery of penicillin and other such antibiotics, people believed that bacteria and bacterial infections would no longer be of concern.Ê The discovery of plasmids inside of various bacteria has proven that belief to be incorrect (Meihong, C. et al. 2005).Ê A bacteria, containing a plasmid, was found to allow us to conduct the experiment.Ê The chance of finding an antibiotic resistant bacterium is much greater in a developed country, due to the common use of antibiotics and antibacterial soaps.Ê After finding and culturing the plasmid containing bacteria, the lyses and purification of the plasmid occured, washing away everything but the plasmid (Krha et. al. 2004.).Ê Restriction enzymes then acted on the plasmid, breaking the DNA up into smaller pieces.Ê The plasmid was then run through gel electrophoresis, showing the number of base pairs the plasmid contains.Ê The banding, when compared to known base pair lengths, gave the information needed to determine the type of plasmid that was being worked with.Ê

ÊÊÊÊÊÊÊÊÊÊÊ Plasmids are extra pieces of DNA that can be found inside of the bacteria.Ê Although this is not part of the bacteriaâs chromosomes, it is a ring of DNA that affects the function of the bacteria by transcribing RNA to later become proteins.Ê The plasmids allow the bacteria to live through various conditions that would generally kill off most other bacteria.Ê For example, the bacteria containing plasmids can survive in harsher environments, such as extreme heat or cold, or in this experiments case, survive through an experience with antibiotics.Ê The plasmid DNA allows the bacteria cell to produce proteins that start degrading the antibiotic when it enters the cell.Ê This therefore kills the antibiotic, not allowing it to function and kill the bacteria (Wikipedia. 2005)

ÊÊÊÊÊÊÊÊÊÊÊ The start of the experiment involved the hunting for bacteria containing these plasmids. When searching for bacteria, I looked in areas that are commonly known, or suspected of having a multitude of bacteria, such as swim bags, bathrooms, trash chutes, and other damp and dark areas.Ê Every swab originally put into the Luria-Burtani contained bacteria.Ê This bacterium was then cultured for several days then added to Luria-Burtani containing Kanamycin.Ê This allowed me to know if the bacteria contained a resistance plasmid.Ê Most of the bacteria we found contained a resistance plasmid to Kanamycin.Ê This shows that antibiotic resistant bacteria are commonly found all over campus.Ê This may be because the University puts antibacterial soaps in all the bathrooms as well as cleans with all antibacterial products.Ê This gives the bacteria a greater chance of obtaining or developing a plasmid.Ê When the bacteria started to grow, they were put on an LB broth containing Kanamycin agar plate, and allowed to grow.Ê When the bacteria continued to grow on the antibiotic containing plate, it was known that the bacteria definitely contained a plasmid. A spread was then performed, putting a small colony of bacteria on a new plate containing Kanamycin.Ê This consists of sterilizing a hook and taking a small portion of the bacteria colony and spreading it over a small edge of the new agar plate.Ê This was followed by sterilizing the hook again and spreading what we already spread onto a different section.Ê After repeating that process several times, you are able to get individual colonies of bacteria.Ê This is the best way to get a single species of plasmid containing bacteria to conduct further experimentation on.Ê The repeat sterilization made sure that no outside bacteria would get into the sample, and potentially alter the results. With only the one strain of bacteria, the bacteria was then culture, or allowed to grow.Ê This allowed a large quantity of bacteria that is known to be resistant to the antibiotics to be collected and further analyzed.Ê By growing a large quantity of bacteria, it allows for a greater amount of plasmid to test and later identify. The bacteria we cultured and grew a large quantity of , was all resistant to the Kanamycin.

ÊÊÊÊÊÊÊÊÊÊÊ After the culturing of the Kanamycin resistant bacteria, the bacteria were run through a series of washes.Ê These washes had different pH levels that would do various things to the cell.Ê The washes purified the bacteria, leaving only the plasmid.Ê This allowed the easy access to the plasmid to be eventually identified.Ê The washes start by breaking down the cell wall, then denature the DNA, then the last wash renatures the plasmid DNA (Krha et. al. 2004).Ê When left with only the plasmid, a restriction digest was able to be run.Ê The restriction enzymes broke apart the DNA every time a particular sequence occurred. ÊThe first attempt at the restriction digest was unsuccessful using Hind III and EcoRI.Ê The digest was most likely unsuccessful due to the fact that neither of the enzymes cut the plasmid DNA.Ê When the Gel Electrophoresis was run, the DNA was apparent in the wells, but did not make any bands.Ê The enzymes successfully used during the second restriction digest were Hind III and Nde I.ÊÊ The enzymes break the DNA into smaller sequences.Ê After we have these smaller sequences, we are able to run the sequences through a gel electrophoresis.Ê The gel electrophoresis showed us, with the use of a known control, the amount of base pairs the plasmid contains. ÊThe gel electrophoresis is able to work on the concept of electric waves.Ê Since DNA is negatively charged, as you send a current through the gel, the DNA would want to move towards the positive end.Ê The chains of DNA therefore travel through the gel and form a line where it is stopped.Ê After a known amount of time, and compared with a known at the same amount of time, the amount of base pairs can be determined.Ê The plasmid that was found in the bathroom sink in 261 East Holmes Hall had length of 3138 base pairs.Ê This data can lead to the belief that the plasmid is pUMVC2, pACYC177, pGPS3, pBR322, and pUC18/19.Ê Those plasmids all have base pairs close to the length determined by the line of best fit, but also only had one of the two enzymes used have any effect on it.Ê The length of the actual plasmid must be looked at within a range between two known base pair lengths that it is between.Ê Since there was only one strand of plasmid DNA, only one of the enzymes did any cutting.Ê That helped limit the list of possible plasmids down to those five.Ê Without the use of more enzymes, it is hard to determine the actual length of the plasmid found in the bacteria.

ÊÊÊÊÊÊÊÊÊÊÊ A common problem that could have arisen during the research is contamination.Ê Since bacteria are so prevalent on earth, it is hard to eliminate all potential of bacteria to enter the experiment at a much later step.Ê This would cause us to work with two different types of plasmids, making the identification very difficult.Ê Although steps were taken to try to work with only one type of bacteria and therefore plasmid, it is impossible to be 100% sure that nothing could have happened.Ê Another problem that could have arisen is human error during procedural steps.Ê The methods used in the research project have a lot of steps; any one missed step could cause a slight variance in the final outcome of the project.Ê For example, the wells created when preparing a gel may have been too deep, or when adding the DNA into the wells, the bottom was pushed through.Ê Both of those problems could have stopped the DNA from traveling through the gel, making it difficult to determine the length of the plasmid.Ê Also, the inaccuracy of measuring the lengths of the bands on the gel can lead to problems when computing the actual length of the unknown.Ê The unknownâs actual length therefore remains in a range, instead of an actual solid number. The major source of error in this experiment came from either misreading the methods or from inexperience with the equipment.Ê Even with inexperience and little accidents, data is still able to be gathered and analyzed to determine the plasmid inside of the bacteria.Ê

(Figure 8.Ê The 1 KB ladder can be seen at the left of the picture.Ê The plasmid DNA sequence that has been cut by the enzymes Hind III and Nde I can be faintly seen, in the middle well, to the right.)