Mutagenesis Lab

This experiment was designed to test and observe the transformation efficacy of the pUC18 and lux plasmids in making E. coli resistant to ampicillin. Both plasmids code for ampicillin resistance, however, the lux plasmid codes for a bioluminescence gene that is expressed if properly introduced into the bacteria’s genome. The E. coli cultures were mixed with a calcium chloride solution and then heat shocked, allowing the plasmids to enter the bacteria and assimilate into the bacterial DNA. The plasmids and the bacteria were then mixed in different test tubes and then evenly spread onto petri dishes using a bacterial spreader, heating the spreader between each sample to make sure there is no cross contamination. Each of the dishes was labeled and then incubated for a period of 24 hours. The results were rather odd because every single one of the samples grew. Several errors could have occurred here, cross contamination or possibly an error in preparation as every single sample in the class grew, meaning all samples of the bacteria transformed and became ampicillin resistant.

Four microcentrifuge tubes were placed in a rack, labeled and numbered, in order to identify the group and the DNA/restriction enzyme that it held. Each of the tubes initially received 10 microliters of reaction buffer. There were two samples of suspect DNA provided along with two restriction enzymes (EcoRI and HindIII). Tubes labeled 1 and 2 received 15 μL of DNA from suspect one while tubes 3 and 4 received 15 μL of DNA from suspect two. Following that, 15 μL of Enzyme 1 (EcoRI) were added to tubes 1 and 3, and 15 μL of Enzyme 2 (HindIII) were added to tubes 2 and 4. (Table 1). The tubes were then gently tapped on the counter to mix the DNA and enzyme solution followed by incubation at 37°C for 45 minutes. After incubation, 5 μL of 10x gel loading dye were added to each of the four tubes of suspect DNA. The tubes were then placed on ice while the gel was under preparation.

The discovery of the gene transfer mechanisms could be attributed by the work of Lederberg and Tatum back in 1946. Using Escherichia coli(E.coli) as their model, they proposed the genetic materia of E.coli could be exhanged via sexual process. In order to prove their hypothesis, they mutated 2 wild type E.coli strains(K12) using X-ray or ultra-violet radiation to produce Y-10 and Y-24 mutant strains. The former was auxotrophic to threonine, leucine and thiamin whereas the latter failed to produce biotin, phenylalanine and cystine[1]. These mutant could only survive in mininal media plates provided that the aforementioned amino acids are supplied accordingly to each mutant strains.

All the procedures performed were according to the Biochemical Techniques: Recombinant DNA Laboratory Second Edition (1). Restriction Digestion of Unknown Vector An unknown vector (0.1 µg/µL) was assigned to the group for restriction enzyme digestions. The vector could be either pUWL 500 or pUWL 501.Three enzymes, Pst I, Bgl II, and Eco RV, and their combinations were used to single, double and triple digest the unknown vector. A total of nine tubes of digestion mixtures were made for this experiment.

Escherichia coli is a well studied prokaryote and may be considered a foundation of biotechnology with the work of Herbert Boyer and Stanley Norman Cohen who used E. coli plasmids in conjunction with restriction enzymes and produced recombinant DNA.1 Even before the application of the rDNA technology, the first industrial application of E. coli being the production of the amino acid threonine in 1961 by the induced mutagenesis of the

Introduction: Mutation within cell populations are seldom.1 The mutS gene in E.coli takes part in the repair and recombination of DNA.2 When mutS was deleted from E.coli in a previous study, the mutation rate increased when compared to the wild type strain.2 Rifampicin is known for its inhibition of RNA Polymerase production.3 Without RNA Polymerase, RNA is incapable of production and thus protein synthesis ceases within the cell, resulting in cell death. We hypothesize that since mutS repairs recombinant DNA within E. coli, the deletion of the mutS gene will increase the mutation rate of E. coli.

The purpose of the experiment was to isolate plasmid DNA, followed by restriction digestion using restriction endonucleases and then visualizing the digested fragments after subjecting to gel electrophoresis. Plasmid DNA (pSP72 DNA) was isolated from Escherichia coli KAM32 (E.coli) cultures using the QIA prep miniprep kit and then subjected to restriction digestion by EcoRI and HindIII. The restriction digested DNA was then loaded into the wells of 0.7% agarose gel and subjected to electrophoresis. It can be concluded from our results that our plasmid DNA isolation was successful and the restriction digestion results were partially in agreement with our hypothesis.

By using a set of techniques that change specific amino acids encoded by a cloned gene, proteins with properties that are better suited than those of naturally occurring counterparts can be created by therapeutic and industrial application. Theoretically, these changes can be made out either the protein or the gene level. However, chemical modifications of proteins are generally harsh, nonspecific, and required repeatedly for each batch of proteins, so it is preferable to manipulate the DNA sequence of a cloned gene to create an altered protein with novel properties.

Restriction enzymes cut DNA at certain sites to create multiple DNA fragments. Restriction enzyme HindIII has known DNA fragment lengths and recognition sites when digesting lambda DNA, while the lambda DNA recognition site for restriction enzyme XhoI is unknown. The goal of this study is to determine the lambda recognition site of XhoI by comparing a HindIII digest and a HindIII and XhoI double digest on an electrophoresis gel. The HindIII digest had a band at 9.4 kb, but this band was not visible in the double digest, therefore we concluded the recognition site for XhoI was around 9.4kb. There were also two additional DNA

At the start of the reversion experiment, 2 drops (0.1 mL) of E.coli B was added to all test tubes labeled B. The same volume (2 drops) of E.coli K was added to test tubes labeled K. All of the test tubes had 0.1 mL of a phage dilution added to their respectively labeled tubes. These test tubes were plated and incubated for 24 hours at 37 ° C and then 4 ° C for 6 days. The next lab period these plates were titered to record how often the mutation reverted to the wild type.

Scientist have harvested many different types of restriction enzymes. Each type of restriction enzyme has a distinct shape and attaches to a specific part of DNA where it eventually makes the cut. Through the use of specific restriction enzymes, scientists can cut out the exact part of the bacteria’s DNA that they would like to use. Once they have a small snippet of DNA from a bacteria that is responsible for what they want to get, like size or taste, they have to get it into the organism. In order to accomplish this a vector is used. The vectors that are used are essentially viruses that have the ability to go into a living thing and insert their own DNA into them. There are other types of vectors besides viruses, but they are the most commonly used. Usually these kind of viruses are very harmful to their hosts, but scientists have removed the harmful part of the virus and instead of it inserted the desired DNA section that they

There was no difference between the control plates which proves that the lid on the plate serves as significant protection, potentially shielding the bacteria to the mutagenic effects the UV light. All three plates were incubated at 25 degree Celsius. The first Control plate showed lots of red and furry growth and the original pattern of swiping was observed in all the quadrants. The second control plate was also observed to have furry and pigmented red growth in all the quadrants, despite being exposed to UV light due to the plate lid during exposure. The experiment plate showed the most interesting results, in the 10 second UV exposure quadrant the bacteria had red pigmented growth but it is speckled, pinpointed and the original swipes were not observed. Moreover the growth seemed not as furry and robust as the control plates. The quadrant exposed to 20 sec of UV light showed significantly less growth than the 10 sec quadrant and the colonies are pinpointed with several beige colonies in between the red speckles. Original swipe pattern of the bacteria was not observed in this colony. In the quadrant exposed to 30 seconds of UV light, the differences are even more significant as the colonies are few and far in between and only several pinpoint red colonies found with a few beige colonies and some colonies with red centers surrounded by beige. The effects of UV light are very drastic in the 40 second quadrant as the growth is severely diminished with only a

This experiment was performed to test the hypothesis if LB nutrient broth, +pGLO and -pGLO Ampicillin, and Arabinose was placed in the E. coli plates, then there will be a significant growth in the newly transformed bacteria and it will possess the ability to glow under UV light. The measurements were recorded from the bent glass tube in each glass test tube. The transformation protocol tested for the newly possessed traits in E.coli bacteria. Throughout the experiment there were many probable reasons for failure. If the pipettes and sterile loop were not thrown out in between each use, a cross contamination could cause a miscalculation in the experiment causing the data results to fail. The hypothesis that was tested was validated due to the positive results with each experiment stating that newly transformed organisms due in fact pass on traits.

The first process used in Gene Therapy is Restriction enzymes. Restriction enzymes are used to locate the nucleotide where the mutation is occurring, as well as cut the viral genome and the new gene, so that both the new gene and the viral genome can attach together at their complimentary base pairs. These enzymes locate specific sequences of bases in order to make either a jagged cut at a specific point on the DNA, called ‘sticky ends’, or

Plasmid DNA with Restriction Digest: The purpose of restriction digest of plasmid DNA is to understand how each DNA plasmids was cut with the given restriction enzymes and perform gel electrophoresis to observe the samples. Nine restriction digests were created, containing three digests for each of the three plasmid DNAs identifying as recombinant, non-recombinant, and unknown. Out of the nine digests, six are actual digests and three are undigested controls. A master mix is created to add to each of the nine samples with its following stock ingredients: 10 ul of 2X Reaction Buffer, 1 ul of Nco1, X ul of sterile water (Single digest), 10 ul of 2X Reaction Buffer, 10 ul plasmid DNA, 1 ul Nco1, 1 ul of Not1, and X ul of sterile water (Double