Living things depend completely on DNA. DNA holds the instructions for the organism’s appearance as well as processes. The survival of an organism in tough circumstances relies on DNA transformation, only when good genes are being used. Viruses, or vectors, are used like taxis to accomplish the transportation of unknown DNA to a host cell. In the experiment conducted in the lab, only the lux plasmid was used, to create a resistance of ampicillin for Escherichia coli cells, and to see if any transformations occurred during the two-week period, where the bacteria was incubated. Two types of growth were expected, either colonial or lawn growth. For the lux plasmid, we knew a transformation had occurred if the agar plate glowed after the two-week period. The agar plates were not incubated in optimal temperature, so at the end of the experiment no transformation had occurred. The results present the question of what is the optimal temperature for lux plasmid. The sterilization process for the agar plates did include some mistakes, the controls for the experiment had some colonial growth due to contamination. Different precautions need to be put into place for future experiments to run smoothly, and result is accurate results, without contamination. Introduction
A collection of amino acids in a molecule that create codes for information that will express certain genes, or DNA. All Populations could be made up of the same genes, some exceptions do exist, small populations sometimes create physical characteristics that unique (Schena, et al., 1995). The central dogma, the process that explains how DNA becomes a protein. DNA will be transcribed into RNA; the RNA will insert all the information that made up the DNA into translational machinery to convert it into proteins. Without the machinery that is used in the translational phase, cells wouldn’t be able to survive. The central dogma is composed of a web of pathways, which are all heavily regulated. The first step of the cellular pathway is the synthesis RNA from a DNA strand, referred to as transcription, as previously mentioned. Translation is when the RNA exits the nucleus and it can be translated into a polypeptide sequence. Primarily this process occurs in both the eukaryotes and prokaryotes, since they differ, the way in which they go about this process differs as well, for the most part researchers can manipulate this process, high volumes of manipulation tends to occur in prokaryotes because they have a more simpler structure and are easier to work with, opposed to eukaryotes (Alberte. et. Al. 2012).
Certain biological systems are improved in humans or plants because of a larger research field, like biotechnology using the practices of some biological systems. In the healthcare field, the production of hormones, vaccines, and antibiotics can all be attributed to the vital role of biotechnology and its practices (Alberte. et. Al. 2012). The process of cell transformation utilizes biotechnology, by which genetic code is manipulated through the insertion of DNA not known to the cell. An application of this, is the creation of a tool that exhibits maximum productivity for metabolic pathways in Aspergillus Niger (Sarkari. et.Al. 2017). Throughout the years, science has caused the ability for humans to transform bacteria into a product which they desire. The transformation can only occur if the bacteria is in a state of vulnerability, a state in which unknown DNA can be introduced without the fear of rejection. In order for bacteria to be vulnerable enough so that it is competent for human intervention, the bacteria must be placed in a substance containing large amounts of a solution known as calcium chloride, which will result in a porous cell membrane. With the cell membrane now having pores, the introduction of unknown DNA will be smoother because the bacteria is now competent. After being placed in the solution, the cells are placed in incubation and heat shocked. Heat shocking the cell with create pores in the bacteria’s plasma membrane allowing the unknown DNA to enter the cell membrane.
Three conditions need to be met in order for transformation to be successful. One, there needs to be a host. Two, there needs to be a way for the host to receive the new DNA, a “taxi”; the “taxi” is known as a vector. Three, a way to identify the modification of the cell must be present, also known as tagging. A plasmid or a virus can be the vector or “taxi”, which is used to transport the DNA into the host cell (Kostina. et. Al. 2017). A circular type of DNA exists in bacterial cells that is set apart from the chromosomes and is known as plasmid (Urray. et. Al. 2016). If a specific circumstance occurs, and a genetic code is desired, this plasmid DNA can clone itself and be passed down to daughter cells. The plasmid will have a marker, this will make it clear that it has been transformed. The lux plasmid is used in the experiment. This plasmid will be utilizing an operon that is found luminescent bacterium Vibrio fischeri, this contains two genes that code for luciferase, and others that code for enzymes which assist in the production luciferins, which will occur in the perfect environment (Godfrey-Smith, 2014). The transformation at the end was produced if the lux plasmid glowed. Escherichia coli still grows when placed together with a plasmid and ampicillin because it has built a resistance.
For the experiment to be performed successfully in the future, four steps need to be completed. First, a vial of Calcium chloride and a tube of Escherichia coli must be placed in an ice bath. A pipet, which has been previously sterilized, will be used to transfer 630 microliters of calcium chloride into a tube containing 50 microliters of Escherichia coli. Mix the solution complete, then let the tube rest on ice for about 10 minutes.
Following the first procedure, the uptake of DNA by cells that are ready will take place. Four tubes are first labeled, the control, control DNA, lux, and plasmid lux DNA, then once all tubes are labeled they are placed on ice. Using a sterile micropipette, 5 microliters of control plasmid will be added to the matching test tube. Make sure the contents of the test tube are will mixed, once that is done, add 70 microliters of competent cells to two tubes. Once again mix the substances of the test tube and put on ice for about 15 minutes. While the test tubes are over ice add 35 microliters of competent cells to the test tubes. Grab another 35 microliters of competent cells and add it to a tube labeled “no plasmid”. Each group must have a test tube labeled “no plasmid” assigned to them. After the 15 minutes are over, transfer the tubes to water that has been preheated to 37 degree Celsius. After this step add 275 microliters of Luna Broth to all control and lux tubes, and add 150 microliters to the NP tubes in all the groups. Once everything has been completed, incubate all tubes at 37 degrees Celsius for 45 minutes. The third part of the experiment will include 6 agar plates in total, divided into two sets of three that will all be labeled properly. Once all agar plates are labeled the test tube substances will be disputed into each plate properly using the proper sterilization techniques. Once all this is done allow the substances to be absorbed, place lid of the agar plate, invert, and incubate at 37 degrees Celsius.
After two weeks have elapsed, the plates are examined for Escherichia coli growth. Each growth is then recorded, as either colonial or lawn. The lux plasmid plates were placed in a dark room to check it they glowed.
After two weeks an incubation period, the results were taken. A total of three photos for Each agar plate was taken because the lab was split into two, to depict any growth that might have occurred over the two week incubation period. One table was of predictions that were made before the incubation of the agar plates, and the second table predicts the results of the experiment conducted in the lab. 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.
The following are the results of the transformation efficiency that was used for finding the transformation efficiency of the LB/Amp with control. The fraction of DNA spread is found by dividing volume of microliter spread of the LB/Amp with control over the total sample volume of microliter in DNA tube. 130/350=13/35. Next, total number of micrograms of DNA is found, and to find that you use the micrograms of DNA times the fraction of DNA spread. Afterwards to calculate the transformation efficiency you need to start by getting the total number of colonies on the LB/Amp with control plasmid plate, divided by the amount of DNA spread on the LB/Amp control plasmid plate. 13 colonies divided by 0.0093 micrograms results in 3.9 x 10 to the power of 3. The same calculations were used to find the transformation efficiency of the LB/Amp with lux plasmid plate. The result was 3.3 x 10 to the power of 3.
Since no ampicillin was present to stop the growth of the bacteria, it was expected that the Luna Broth with the control plasmid would be the plate with either colonial or lawn growth. Alongside the Luna Broth with control plasmid, it was predicted that it would also grow because of the introduction of plasmid. As a lab, the possibility Luna Broth with no plasmid would grow because ampicillin was not present was also known. Bacteria would be terminated by ampicillin if plasmid is not present in the agar plate, because of this LB/Amp with no plasmid was not predicted to having any type of growth. As expected and tested, any agar plate with plasmid will grow, excluding the presence or absence of ampicillin. Resulting in both LB/Amp and LB with lux plasmid to have either colonial or lawn growth in the agar plate. Since lux is present in the plasmid, agar plates with lux were expected to grow, also indicating that a transformation had occur while the plates were in incubation for the two-week period.
The results of the lab were increasingly consistent throughout the lab, in terms of all the predictions that were made. Once observations were made, it was concluded that Luna Broth with control plasmid had a lawn growth because of the absence of ampicillin. Colonial growth was recorded for Luna Broth with ampicillin and the control plasmid because the ampicillin didn’t stop the growth but it did attempt to prevent any growth; same results were recorded with Luna Broth with ampicillin and the lux plasmid. The agar plates with the most growth also known as lawn growth were Luna Broth with no plasmid and Luna Broth with lux plasmid, because no ampicillin was present and the bacteria can grow freely and exponentially. The colonial growth that occurred with Luna Broth with ampicillin and no plasmid was not expected and should not have happened, because there was ampicillin meaning bacteria should have been killed off. This lead to the conclusion that the sterilization techniques taken throughout the experiment were not up to par with what should have occurred. The only explanation the growth is contamination of the agar plates before the two-week incubation period. The main reason that the lux plasmid agar plates did not glow was because the lux operon are enzymes that are only activated under optimal temperature which was present during this experiment; if the lux optimal temperature were achieved than the plates would have glowed indicating a transformation had happened. For future experiment, two things need to be taken into consideration before beginning. First, new sterilization techniques need to be implemented and tested to ensure the accuracy of the experiment. Second, the optimal temperature of the lux operon needs to be known and the experiment needs to be adjusted to suit the needs of the lux operon to better the results of the experiment. As for the efficiency of the transformations, the number of colonies in the agar plates and the number of microliters given, are two factors that could have affected the results.
I accept the hypothesis because there was a relationship between cell growth and plasmid which was the prediction made. Results indicate that once plasmid is introduced to a bacteria, growth will be allowed, even in the presence of an antibiotic like ampicillin.