SDSU Biology Lab Report
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When the process of DNA cloning was discovered it revolutionised the study of molecular biology. It enabled scientists to select a specific part of a DNA sequence and to produce it in an unlimited amount by placing the DNA fragment within a plasmid shuttle, or vector. DNA cloning has allowed scientists to amplify DNA which has a variety of applications in genetics. This includes being able to study its sequence to better understand how DNA codes for traits, it gives scientists the opportunity to manipulate and engineer the DNA, and to use it for expression and generation of protein (Sadava et al., 2009). For example, the discovery of DNA cloning allowed the mass production of the hormone insulin for diabetes which has saved countless lives (Goeddel, 1979). The ability to generate protein has also proved to be particularly useful and can be used to produce antibiotics such as the bactericidal antibiotic gentamicin, which is the focus of this paper.
There were several discoveries which enabled us to understand DNA cloning as we do now. In 1944, Avery, Macleod, and McCarty demonstrated gene transfer was possible using isolated DNA from the bacteria S. pneumoniae, this showed it is possible to obtain fragments of DNA. In 1968 by Arber and Linn, discovered an enzyme which could selectively cut exogenous DNA, which provided the mechanism to incorporate a DNA fragment into a plasmid in order to amplify it. They called this enzyme a restriction factor (Quail, 2010).
Restriction enzymes are crucial in DNA cloning as they are needed to digest the DNA sample and plasmid vector. The same enzyme is used for the plasmid and DNA in order for the restriction sites to match and ensure successful ligation. This is achieved by finding a palindrome sequence (sections of DNA exhibiting two-fold symmetry) for the enzyme (Lodish et al., 2000). To increase the chances of ligation restriction enzymes usually leave overhangs, for example Bam HI leaves a 5’ overhang and Kpnl leaves a 3’ overhang (Quail, 2001). In this experiment, a type II restriction enzyme called HindIII was used (Lab Manual, 2020). Type II enzymes are distinguished from other types of restriction enzymes by the ability to cleave DNA at specific locations within the restriction site. Then, DNA can combine with the plasmid and the enzyme DNA ligase joins the DNA and plasmid together in a condensation reaction creating phosphodiester bonds and water as a by-product. To improve the chances of ligation alkaline phosphate is used to remove 5’ phosphate groups from DNA to prevent self-ligation.
Once the plasmid is inserted into E. coil, the bacterium must be grown under restrictive conditions using an antibiotic growth medium, as the chances of successful recombination is small. Wild-type E.Coli cultures do not contain the gentamicin resistance gene. Therefore, to select for the recombinant plasmid the E. coil bacteria can be allowed to grow for around an hour in a growth-rich medium, to allow the gene for antibiotic resistance of gentamicin to be replicated. Then, when the E. coil is plated on agar with gentamicin, only those that have successfully taken up the ligated vector will survive.
The purpose of this study was to insert a DNA sequence coding for gentamicin into the bacterium E. Coli by using the Hind III restriction enzyme to digest the plasmid creating a site for the DNA, then running the fragments of the cut plasmid on a agarose gel to sequence the correct plasmid. Gel electrophoresis was used to determine the digested products of the restriction enzyme. Overall this study outlines the full process of cloning DNA and inserting into a bacterium.
Material and methods-
Exercise 3 of the lab manual on Basic cloning methods involved the following experimental procedures: amplification as well as purification of plasmid vector, litigation and purification of litigation, digestion of restriction enzyme, gel electrophoresis as well as electroporation. All these were carried out as indicated on pages 37 through 49. In part B step 5, N4 solution was added and the result was formation of a somewhat cloud mixture. When carrying out electroporation, the machine failed to beep. This in turn caused the plate to have no growth.
Exercise 4 on “Cloning Verification”, was detailed on pages 51 through 59 of the lab manual. The procedures involved transformant plasmid vectors, sequencing of insert, and gel electrophoresis. All the procedures were followed as they were laid out on the laboratory manual. Exercise 4 was more involving for the group compared to exercise 3 and 5. In our group, all the members understood and followed the experimental procedures without any major challenges. The results were obtained as expected. Exercise 5 on “Bioinformatics” entailed the procedure of genetically mapping the plasmid resistant to gentamicin using ApE software. This procedure was conducted as detailed on pages 61 through 62 of the laboratory manual (Lab manual, 2020).
All the experiments were connected in some way. One step in the procedure provided a basis for the next step. That is why it was necessary for the steps to be followed exactly as outlined in the manual. Throughout the three exercises, we were keen on noting the results of each procedure and noting the relation to the expected results of the experiment. The results obtained from the experimental procedures are shown in the figures below. Each figure is labelled and an explanation given as to what it entails in connection to the experiments.
Fig 1: streak plate of LB agar and gentamicin
Fig1 this shows an illustration of the outcome from exercise 4. There were no isolated colonies formed as one can see.
Fig 2: Gel electrophoresis after PCR
Figure2 shows the results for group 1 after we ran the gel electrophoresis. The figure was observed under a Trans illuminator. Looking at the image, we found no bands formed.
Fig 3: Gel electrophoresis of restriction digest
Fig 3 shows the results of exercise 4 where gel electrophoresis was done and observation made on the formation of bands. As seen from the figure, there was band formation.
The experiment involved three exercises; exercises 3, 4, and 5 detailed on the laboratory manual. The aim of experiment 3 was to perform basic cloning methods. The plate from exercise 3 was used in exercise 4 to investigate the colonies formed. As seen from the figure 1 illustrated in the results section above, there were no isolated colonies observed. This phenomenon resulted from the failure of the electroporation machine. The machine was supposed to beep to indicate electroporation to happen but did not beep. This resulted in a lack of growth on the plate and this explains why no isolated colonies were formed.
After the polymerase chain reaction (PCR) gel electrophoresis was done as outlined in the exercise 4 procedure. The main purpose of conducting the gel electrophoresis was to separate the products of restriction enzyme digest and observe the results. In our group, we realised that no bands formed the electrophoresis. This result shows that there was
Failure on the part of PCR. The polymerase chain reaction was not successful. The probable cause of this failure is a malfunction of the master mix.
When gel electrophoresis of the restriction digest was performed, we observed that some bands were formed. This gives an indication that the procedure was successful. Looking at the results, it is clear that bands were formed for both the digested and undigested wells. In conclusion, the experiment was a success. The main challenges we faced, and which altered the results of our experiment are: failure of the electroporation machine and an unsuccessful PCR resulting from a malfunction of the master mix.
Quail, Michael Andrew. (2001) “DNA cloning”. The Wellcome Trust Sanger institute
Goeddel, David V., et al. (1979) “Expression in Escherichia coli of chemically synthesized genes for human insulin.” Proceedings of the National Academy of Sciences 76.1 (1979): 106-110.
Sadava, David E., et al. (2009) Life: the science of biology. Vol. 2. Macmillan, 2009.
Lodish, Harvey, et al. (2000) “Molecular cell biology 4th edition.” National Center for Biotechnology Information, Bookshelf
all part need fixing and adding except the introduction
I will provide an example from other student
try to provide the same amount of information provided in the example but based on my result
but go off of what they wrote but make sure to not copy anything
take the idea and write it in your own words
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