Abstract
The vitality of life and several biotech industries depend on DNA. DNA can be used in advancing cloning methods. DNA transformation with the use of good genes, can be the factor of the survival in harsh conditions. Plasmids/viruses, or vectors, are used to accomplish the transportation of foreign DNA into the host cells. The experiment used two plasmids, pUC18 and lux plasmid, to try to achieve resistance of ampicillin for Escherichia coli cells, and determine if the cells were transformed during a two-week period of incubation. For the plasmids, we expected either colonial growth or lawn growth. For pUC18 colonial growth was expected, meaning that if there was colonial growth transformation occurred. As for the lux plasmid if the agar plate glowed, that would mean that transformation occurred. In the experiment, the lux plasmid did not glow due to the plasmid not being incubated in optimal temperature. This would beg the question what temperature would be best for the lux plasmid to glow. As for the pUC18 colonial growth occurred, this means that the plasmid achieved the end goal of the experiment, and that transformation occurred. As for the controls, there was some contamination that resulted in colonial growth, therefore the aseptic techniques were in some way flawed, and raises the question of what should be done for future experiments to ensure that no contamination occurs.
Introduction
DNA is a molecule that has a collection of amino acids that codes for information that will express certain genes. Each population could have the same genes, with certain exceptions of a small population that could produce physical characteristics that are unique (Schena, et al., 1995). DNA is transcribed into RNA, and RNA will then go to the necessary translational machinery to convert, the information of DNA, into proteins. The machinery that allows for transcription to happen from DNA to protein is extremely important for life. The process of this, involves a web of pathways, and each pathway is regulated. The synthesis of RNA from a DNA template is the first step of the cellular pathway, and is referred as transcription. When the RNA exits the nucleus than the RNA can be translated into polypeptide sequence, this is referred as translation. For the most part, these pathways take place in both eukaryotes and prokaryotes, and each having their own respective way of how to do each, but researchers can manipulate this process, and prokaryotes are used the most in this manipulation, due to the fact that they are easier to work with than eukaryotes (Alberte. et. Al. 2012)
As a relation to a larger research field, biotechnology uses the application of certain
biological system to make certain products or improve certain biological systems of humans, and or plants. Such applications of biotech have played an important role in producing hormones, antibiotics, and vaccines, in the healthcare system (Alberte. et.Al.2012). Biotech is used in the process of transformation of cells, manipulating the genetic code, through the input of foreign DNA. One example is creating an efficient tool for metabolic pathways for Aspergillus niger (Sarkari. et.Al. 2017) Humans have intervened to transform bacteria, into a desired product or effect. For this to happen though, the bacteria must be in a state of competency, in where the foreign DNA can be introduced. For the bacteria to be reached to state of competency, the cell membrane must be porous, a way for this happen is to the place the bacteria into a solution with a large amount of calcium chloride. This will help the cell membrane to have pores in where it increases the cell competency, the cell’s ability to pick up the foreign DNA. Afterwards the cells are put in incubation and then heat shocked, this process of heat shocking will create pores in the plasma membrane of the bacteria and allow the foreign DNA to enter the cell membrane.
For the transformation to succeed, certain conditions need to be met. First, there must be
a host; secondly, something needs to transport the DNA into the host, this “something,” is called a vector; and thirdly a method of identification, or known as tagging, to make sure if the cell was modified. The vector, which is used to transport the foreign DNA, can be a plasmid or a virus (Kostina. et. Al. 2017). In the nucleoid region of bacterial cells, a small circular type of DNA exists apart from the chromosomes, and this type of DNA is known as plasmid (Urray. et. Al. 2016). This plasmid DNA can be replicated and can be passed along to daughter cells to express the desired genetic code, if the situation arises. The plasmid will have a gene that serves as a way to distinguish transformed cells, and this is called a marker. The plasmids used in the experiment are pUC18 and the lux plasmid. The plasmid pUC18 contains an ampicillin- resistant gene that enables certain bacteria to grow in the presence of antibiotics. As for the lux plasmid, the lux that we will be using is an operon that is found in the luminescent bacterium Vibrio fischeri, this bacterium contains two genes that code for luciferase, and several other genes that code for enzymes that will produce luciferins if the temperature is optimal (Godfrey-Smith, 2014). The results of transformation when the lab was produced contained was whether the lux plasmid glowed, and if the pUC18 created bacteria that was resistant to ampicillin, when it was incubated. We saw that if you place E. coli, with a plasmid, on a medium with ampicillin, then the bacteria will still grow because it is ampicillin resistant.
Methods
For the methods, there is four procedures that must be followed in the experiment for it to be able to be replicated. For the first part of the procedure is where we will prepare the competent cells. First, a vial with CaCL2 solution and a tube of E. coli must be placed in an ice bath. A sterile pipet will be used to transfer 590 microliters of the calcium chloride solution to a tube containing 50 microliters of bacteria. Mix solution with a tap, and then incubate the cells for around 10 minutes on ice.
For the second part of the procedure, it is where the uptake of DNA by competent cells will take place. The control, control plasmid DNA, and the lux, plasmid lux DNA, are labeled and then placed in an ice bath. Use a sterile micropipette, and add 5 microliters of control plasmid to the tube labeled for it. Tap the tube gently to make sure all the competent cells are in suspension, afterwards add 70 microliters of the competent cells to the two tubes. Mix the two solutions with a tap of a finger on the tip of the tube, and then place them in ice for 15 minutes. While the solutions are in the ice bath, add 35 microliters of competent cells to each tube. Obtain an additional tube with the 35 microliters, and label the tubes, “no plasmid.” Each group has to have NP tube assigned to them.
After the 15 minutes are up, transfer the tubes to a water bath preheated to 37 degrees Celsius for 5 minutes. Afterwards a sterile pipet will be used to add 275 microliters of nutrient broth to the control and lux tubes. To the NP tube add 150 microliters of the nutrient broth. Once everything is ready tubes must be incubated at 37 degrees Celsius for 45 minutes.
For third part of the procedure, you will gather 6 agar plates, 3 for each group, and label them respectively. Afterwards, get 130 microliters of the mixed bacterial suspension from the control tube and dispense the bacteria onto the control agar plate. Spread the bacteria using a cell spreader evenly across the plate. Once the bacteria are mixed evenly, again use the cell spreader and dip it in ethanol, and pass the spreader through the flame of an ethanol lamp. Afterwards transfer 130 bacterial suspension from the “lux” tube to its respective agar plate and spread the cells on it. Repeat the process of cell spreading with the cells that contain no plasmid onto their respective plates. Replace all the lids of the agar plates, with new ones and let the plates absorb the liquid at room temperature. Once that has been done invert, and incubate the agar plates at 37 degrees Celsius.
After two weeks of incubation, the plates are examined and checked to see if the E. coli growth occurred. Each growth type was recorded, as either colonial or lawn. The lux plates were put in a dark space to check to see if bioluminescence occurred.
Results
After two weeks an incubation period, the results were taken. A total of six photos for
each agar plate was taken to show the type of growth that was resulted and two tables were made. One table is for the predictions that were made two weeks before the incubation period, and the other table is the results. The results show the type of observed growths occurred, whether or not it was bioluminescent, and the reasons for the observed results. The table of predictions shows the expected type of growth and if it will be bioluminescent, and the reasons for why we expected those predictions.
In the results, we made a table to predict what would happen to each of the agar plate.