Dna Ligation Lab Report

What was expected that the strains of E. coli that did not have a resistance to ampicillin would not grow. The transformed strain also changed to a blue color when the X-gal was present in plate. The transformed cells also grew because they were free of the ampicillin because they possessed the amp gene that they used as an shield against the ampicillin antibiotic. The transformed cell who turned blue did so because the gene converted the sugar to a blue color but also contained the amp gene to ensure that they grew even when the ampicillin was present. The growth of the colonies on the plates

The color of the bacteria was a whitish color and the colony size is similar both before and after the transformation. The best way to do it is to compare the control of the experimental plates. Cells that were typically not treated with the plasmid could not grow on ampicillin, although cells that were treated with the plasmid can grow on the LB/AMP plate. The plasmid would have to confer resistance to ampicillin. Moving on, the GFP gene is what is glowing in the plate because it was activated by the sugar arabinose. The sugar arabinose and the plasmid DNA are also needed to be present because that is what initially turns on the GFP gene which makes the bacteria glow. Organisms can also turn on and off particular genes for camouflage reasons. An organism would benefit from turning on and off certain

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.

70µL of competent E.coli are added to both test tubes; pUC18 and Lux (Alberte et al., 2012). Both test tubes are then tapped and placed back into the ice bath for 15 minutes. While waiting, another test tube is obtained, filled with 35µL of competent cells and labeled NP for no plasmid. A water bath is preheated to 37 degrees Celsius and all three labeled test tubes are inserted into the bath for five minutes (Alberte et al., 2012). Using a sterile pipet 300µL of nutrient broth are inserted into both the control and Lux test tubes and 150µL are inserted to the no plasmid test tube to increase bacterial growth. All three test tubes are then incubated at 37 degrees for 45 minutes. Six agar plates are obtained and labeled to correspond each test tube, three of the plates contain ampicillin. A pipet is used to remove 130µl from each test tube containing a plasmid and insert it into the corresponding agar plate. For this, a cell spreader is first

The plasmid pGLO contains an antibiotic-resistance gene, ampR, and the GFP gene is regulated by the control region of the ara operon. Ampicillin is an antibiotic that kills E. coli, so if E. coli, so if E. coli cells contain the ampicillin-resistance gene, the cells can survive exposure to ampicillin since the ampicillin-resistance gene encodes an enzyme that inactivates the antibiotic. Thus, transformed E. coli cells containing ampicillin-resistance plasmids can easily be selected simply growing the bacteria in the presence of ampicillin-only the transformed cells survive. The ara control region regulates GFP expression by the addition of arabinose, so the GFP gene can be turned on and

The bacteria has ampicillin resistance gene and can either have no RNAi insert (control plate) or PCR product that is a portion of target gene (unc-22).

The purpose of this experiment is to make E.Coli competent so that it can be transformed in order to become immune to ampicillin, then we would be able to determine the transformation efficiency of the culture. We determine this by preparing 4 plates of E.coli, each labeled “LB-plasmid”, “LB+plasmid”, “LB?Amp-plasmid”, and “LB/Amp+plasmid”. This meant that either should have lacked plasmid and Ampicillin, with plasmid but lacked Ampicillin, without plasmid but with Ampicillin, or were with Ampicillin and plasmid, respectively. Then we made the bacterial cells competent by adding CaCl2 to 2 vials of the colony (one with plasmids), and incubating on ice, then heat shocking, and returning to ice. Luria Broth is then added and left to sit for 5-15

The purpose of this experiment was to show the genetic transformation of E. coli bacteria with a plasmid that codes for Green Fluorescent Protein (GFP) and contains a gene regulatory system that confers ampicillin resistance. A plasmid is a genetic structure in a cell that can replicate independently of chromosomes. In this lab, the Green Fluorescent Protein, which is typically found in the bioluminescent jellyfish Aequorea Victoria, was cloned, purified, and moved from one organism to another with the use of pGlo plasmids. It was hypothesized that if bacteria that were transformed with +pGlo plasmids are given the gene for GFP, then transformed cell colonies

The pGLO plasmid will transform the E. coli bacteria with a gene called GFP that codes for the Green Fluorescent Protein in the genetic code. GFP was discovered in the jellyfish, Aequorea victoria as a green fluorescent light emitted from the jellyfish. It was typically seen in the dark upon its activation and since then has been used in studies relating to genetic transformation. (Chalfie and Tu 1994) The majority of the studies test the many different factors that are required in the transformation of pGLO which will determine the functionality of GFP in the E.coli bacterium. The first experiment in transforming GFP and E. coli was completed in 1994 by Chalfie and was further refined the same year. The experiment proved the importance of using restriction enzymes, and DNA ligase in the process of transforming GFP to identify arabinose as the primary activator, and to identify the ampicillin

For this experiment, E. coli was best for genetic engineering because of their size, and their fast reproduction (Spilios, 2017). E. coli will be genetically transformed using an engineered plasmid. A plasmid is a circular piece of DNA which independently replicates and multiplies because it has its own origin of replication (Spilios, 2017). The pGLO is the plasmid used in this experiment. Plasmids are used as vectors and they contain manipulated genes such as genes coding for antibiotic resistance for drugs like ampicillin. This antibiotic resistance of such serves as the selectable marker in genetic transformation and for genetic transformation to proceed, the cell must reach competency which is the physiological state that is required for the vector plasmid to get into the cell for transformation (Spilios, 2017). While competency can be reached naturally in some organism, it must be reached artificially in E. coli through treatment with CaCl2 and exposing them to heat shock using incubation (Spilios, 2017).

While the plate with -pGLO on the LB/AMP agar plate would not have any colonies, and the –pGLO on the LB agar plate would have many colonies of E. coli. The +pGLO on the LB/AMP plate did have two colonies but the only colonies produced on the LB/AMP/ARA plate had a colony not growing on the actual agar plate. The –pGLO on LB/AMP did not have colonies of E. coli growing on it. But the –pGLO on the LB agar plate had many colonies growing on the plate. All of the colonies were clear in color even under the ultraviolet light. Therefore the plate that did not support my hypothesis was the plate with the arabinose sugar, since that was the one thought to grow E. coli do to the ampicillin resistant DNA that was in theory picked up by the E. coli. This plate also should have glowed green under the UV light but instead the colonies remained clear. I hypothesize that E. coli cells recognized the foreign DNA and then destroyed it believing the new DNA would be harmful to the cell. This tendency of bacteria cells to kill foreign DNA could answer be a problem that would explain why we had few colonies that survived in the presence of ampicillin and why none of the colonies in the arabinose glowed green. Also the time the E. coli

Plasmid map of pRSETB. Primers were designed to amplify via PCR to cDNA. The PCR product was digested with XhoI and EcoRI enzymes and ligated into the pRSETB plasmid. The pRSETB plasmid contains a T7 promoter region, is ampicillin resistant, inducible with Isopropyl β-D-1-thiogalactopyranoside IPTG, a molecular mimic of a lactose metabolite that triggers transcription of the lac operon, and has XhoI and EcoRI cut sites. The pRSETB plasmid is transformed into dH5α E. coli and plated on carbenicillin plates. Colonies are selected and grown on a carbenicillin plate while PCR is used to check that the plasmid that was up-taken was not

This two-week lab had an overall theme of DNA analysis. Humans can distinguish between five different tastes: sour, sweet, salty, bitter and umami. In the 1930’s, a man named Arthur Fox created a molecule that some people can taste and others cannot, it’s called phenylthiocarbamide or PTC. It is based off a gene variation, whether or not someone can taste it. Tasting PTC is dominant and not tasting it is a recessive gene. The TAS2R38 gene can also be shown as incomplete dominance which means that the person can barely taste it. This can be tested by collecting a sample of cells from the human mouth and extracting the DNA by boiling it with a Chelex resin. This binds to metal ions to help

The mutant β-galactosidase is unable to form a tetramer and is inactivated. When the recombinant vector containing N-terminal fragment of the protein, the α-peptide are expressed together with ω-peptide, they form a functional β-galactosidase enzyme. The blue white screening works by disrupting this α-complementation process. White colony on X-gal indicate the present of foreign DNA fragment in lac Z gene of plasmid. Hydrolysis does not take place on X-Gal because the insertion disrupt the formation of active β-galactosidase. In contrary, blue colony indicate the absent of foreign DNA fragment in lac Z gene of plasmid. This is because functional β-galactosidase enzymes are produced to hydrolysed X-Gal, create a blue

The domestication of animals 30,000 years ago was the precursor to Genetic Engineering. Starting with the dog we have since been able to introduce desirable traits in all organisms. With the discovery of plasmids in the late 60’s we have been able to take genetic engineering even further. Plasmids are small circular DNA molecules used to amplify and replicate a gene of interest. These minute molecules have the ability to replicate with the chromosome or independently, allowing them to have up to a 100 copies in one cell. Plasmids are important because of their characteristics to transfer genes that occur naturally within them or acting as a vector to introduce foreign DNA into a host cell. With the use of plasmids genetic engineers are able to use bacterial transformation to make medicines. Bacterial transformation is the process in which a bacterial cell takes up foreign DNA and incorporates this DNA into its own. With the use bacterial transformation this part of Genetic Engineering has become the most important and widely used technique, creating life saving antibiotics and medicines. E.coli is generally used in these procedures because of its ability to adapt and grow exponentially. Genetic Engineers can then use large bacterial colonies, Ampicillin, and X-gal to indicate if b-glactosidase is present along with identifying the recombinant and non-recombinant colonies. The transformed bacteria that contains ampicillin will spread and survive either turning white