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.

Genetic Transformation of E. coli Using pGLO Plasmid Introduction The bacteria E. coli is a competent bacteria which has the ability to accept foreign pieces of DNA and express them in itself. In this lab will be testing the hypothesis that E. coli is competent and can express foreign DNA by depositing pGLO DNA, which was created from the same DNA that makes jellyfish fluorescent, into the E. coli to make the bacterium glow. We are also testing the hypothesis that the pGLO DNA can make the E. coli resistant to ampicillin.

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.

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

2. Zion, Michal, et al. "UV Radiation Damage And Bacterial DNA Repair Systems." Journal Of Biological Education (Society of Biology) 41.1 (2006): 30-33. Academic Search Complete. Web 15 Oct. 2014.

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.

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.

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.

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.

As a result, in comparison to point mutation, frameshift mutation has been showing strong reaction to the mutagens. As a control, deletion mutation plate has showed all purple wells, indicating there is deletion in the histidine synthesize. Since it was not reversed into wild it showed purple color. As for the second control bacteria without mutagen, since it has been already either point mutated or frameshifted, the color was still purple.

(1A) Design a forward genetic mutagenesis screen, in a fly or rodent model, that would allow you to identify genes required for proper axon guidance. Discuss the main steps involved in this approach.

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

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

Site-directed mutagenesis, also known as oligonucleotide-directed mutagenesis, is a technique that is used to intentionally modify a DNA sequence of a gene by introducing a site-specific mutation. The general method for producing the defined oligonucleotide mutation was developed from a combination of observations on nucleic acids, and it was in the 1978s where enzymatic extension was used as an approach to SDM, using oligonucleotides as primer (Hutchinson, et al., 1978). Since then, this technique has then been improved in efficiency in later studies which has stemmed from this initial mutagenesis experiment, and thus novel methods of inducing site-specific mutagenesis has been improved in later years.

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