Quality Report on pGLO prep Genetic transformation is a process that modifies bacteria, by introducing new genetic material. In our lab we introduced the pre-engineered pGLO into the E. Coli HB101 K-12 bacteria. This pGLO plasmid consists of the gene for the green fluorescent protein (GFP), the ampicillin resistance gene that inactivates the ampicillin in the LB media and the araC gene that indirectly controls the arabinose digestion enzymes [Fig 1]. Green Fluorescent Protein fluoresces bright green when exposed to UV light. The GFP gene only activates if there is arabinose present. When arabinose is not present, the arabinose digestion genes are inactive and energy will be conserved. However, when arabinose is present the genes activate and start to break down the arabinose until it is all consumed. BAD encodes for the enzymes used to digest arabinose. The araC gene in the DNA map above pairs with arabinose, and up regulates BAD. However, it also negatively feeds back into its own regulation. Bla is the Ampicillin Resistance gene produces Beta lactamase, a protein that confers ampicillin resistance [1]. In our lab, we used chemically competent cells, which are bacteria cells that …show more content…
A ratio of approximately 1.8 is considered pure for DNA. If the ratio is lower than 1.8, it indicates some contamination. This contamination can be attributed to the presence of protein. Another measure of purity is the ratio of absorbance at 260 and 230nm. This ratio is generally slightly higher than the 260/280 ratio and a ratio of approximately 2.0-2.2 is considered to be pure. In this case, if the ratio is lower than 2.0, it indicates contamination [3]. Our Nano-drop data after we purified the plasmid came out to be 1.91 and 1.42 respectively. Our 260/230 ratio was significantly lower than our 260/280 ratio that signifies that there was some other contamination in our
Once the recombinant plasmid was obtained, it was then inserted into E. coli cells through transformation. From a successful transformation, we expected the bacterial cells to translate the inserted EGFP sequence into its protein form. The bacteria cultures were plated on petri dishes containing growth supplement, Luria Broth (LB), an antibiotic: Kanamycin, and IPTG which induced the fluorescence property within successfully transformed bacterial colonies. Different variants of the petri dishes were also included as control and unknown.
Serratia marcescens, a Gram-negative bacillus, was originally and solely considered a biological marker in the medicinal industry, due to its highly natural red pigment: Prodigiosin (Hejazi and Falkiner, 1997). The pigment has numerous roles within bacteria, which can be further translated into the pharmaceutical and medical domain. This bacterium naturally occurs in water, soil, on plants as well as in humans and animals (Khanafari et al, 2006), where it is deemed an opportunistic pathogen.
al. (1994) explain that a complementary DNA for GFP produces a fluorescent product when expressed in E. coli cells as the expression of GFP can be used to monitor gene expression and protein localization in living things. In this experiment, the heat shock method will be used to deliver a vector (plasmid) of GFP to transform and grow E. coli bacteria. Four plates containing Luria Bertani (LB) broth and either –pGLO or +pGLO will have E. coli bacteria added to it. The plate containing –pGLO (no pGLO) and LB will show growth as ampicillin will be present killing bacteria but no glowing because no arabinose will be present for glowing to be activated, the same result will be seen in the plate containing +pGLO, LB and ampicillin.
The main goal for our experiment was to learn how to examine DNA when there is only a small
The expression of lac operon in each tube equals the amount of beta-galactosidase produced. Therefore, by looking at the amount of beta-galactosidase under different conditions collectively is a good way to understand the function of inducers and repressors in supervising the expression of lac operon and the control of gene expression generally.
...et light. If the LAA plate glows green under exposure to ultraviolet light, then we can conclude that our unknown insert piece of DNA would be the kan gene. If it does not glow green under exposure to ultraviolet light, then then we streak the colony from our LAA plate onto the LAC plate using a sterile glass spreader. When the LAC plate is dray, we place it upside down in the microfuge rack so that it can be incubated at 37 ºC. Incubation at 37 ºC will allow the transformed bacterial cells to grow. If we see bacterial growth on the LA plate containing chloramphenicol, we can conclude that our unknown insert piece of DNA would be the cat gene, since the cat gene is resistant to chloramphenicol. Afterwards, we then grab the microfuge tube labeled NP and repeat the aforementioned steps shown above pertaining to the LA plates. This would be considered our control.
I can do to verify its purity by viewing colonial morphology. I will streaking this culture on the plate by using streak plate method and viewing their cellular morphology by preparing a smear. I also view it by using a microscope which it will be 1000x. If the culture was not pure, I would notice the streak plate may have colonies with different colonial morphology, color, and cells. I have to view it in the microscope so it may have different cellular morphology and staining properties. The five ways I can contaminate a culture during inoculation are accidentally touching the loop or needle, not carefully holding the broth and leave it on the table so it will leaking down the tube, working in the lab that have a lot of airborne contaminants which it is mixtures exposure by absorption through skin, a sample open for a long time, and forget to flame the needle, loop, or tube.
A Ponceau stain can bind and identify all proteins. Lanes 2, 3, and 4 (our recombinant, nonrecombinant and green colony, respectively) have a slightly smeared pattern of multiple bands that goes from 245 kDa to 80 kDa. Lanes 2 and 4 have faint banding patterns that descend from 80 kDa downwards. Lane 3 ends a bit early, around the 135 kDa mark. Lanes 5-7 (our white colony, unknown colony and purified
Antibiotics have the ability to kill or hinder the growth of bacteria. Antibiotics contain compounds that are naturally produced by organisms to combat diseases caused by microbes. Discovery of penicillin by Sir Alexander Fleming became the first stepping stone of many new antibiotics of today’s modern medicine. Antibiotics typically invade the very components that make up bacteria, such as cell walls and metabolic pathways (Sato et al., 2014). However, frequent mutations of bacteria cause today’s strains to become more resistant. One of many ways which bacteria undergo mutation is through horizontal transfer of genes (Lindsay J.A., 2013). The war against disease is a battle that humanity has fought for centuries, and only recently has the development of penicillin switched that tide of war in our favor. However, with the advent of methicillin resistant staphylococcus aureus and even vancomycin resistant staphylococcus aureus, the prospect of this battle is not promising (Bobenchik et al., 2013). Thus, it is crucial to test bacteria for antibiotic resistance to utilize antibiotics that battle with bacteria properly.
...rm (1:1) was added to the linearized sample in a 1.5 ml microcentrifuged tube. The mixtures were centrifuged at 13000 rpm for 2 minutes at room temperature. The upper aqueous solution was transferred to another sterile 1.5 ml microcentrifuged tube. Equal volume of chloroform was added and centrifuged at 13000 rpm for 5 minutes. Again, the aqueous solution was transferred to a new 1.5 ml microcentrifuged tube and 1/10 volume of 3M Sodium Acetate Solution was added. Then, 2.5 volumes of cold absoluted ethanol were added to precipitate the DNA. The mixture was incubated in -20 °C for overnight or -80 °C for 1-2 hour.
Ampicillin disrupts the third and final stage of bacterial cell wall synthesis by binding specific penicillin-binding proteins (PBPs) that are inside the bacterial cell wall. Then facilitated by bacterial cell wall autolytic enzymes, cell lysis beings. Ampicillin is metabolized by Hydrolysis of the B-lactam ring to penicilloic acid. Microorganisms such as salmonella, Escherichia coli, campylobacter, shigella aquificae, thermotogae, chrysiogenetes, nitrospira, deferribacteres, other eubacteria, and other enteric bacteria are sensitive to Ampicillin. Treatment Dosage can range from 1 to 2g IM or IV every 4 to 6 hours to the maximum does of 12g per day. Microorganisms resistant to Ampicillin are penicillinase- producing bacteria (some strains of staphylococci), Pseudomonas aeruginosa, P. Vulgaris, Kiebsiella pneumonia, and Enerobacter aerogenes.
SAN MIGULE ARCANGEL The San Miguel Arcangel is unique among the twenty one Spanish missions of California. San Miguel Arcangel was the sixteen of twenty one missions and there by shorten the long distance between the San Antoino and San Luis Obispo missions. In 1806, many of the mission building and all of the supplies destroyed by fire. Mission San Miguel Arcangel is named after Saint Michael the Archargel.
This type of resistance may be at a high level. This mechanism of resistance is mediated by the erm (Erythromycin Resistance Methylase) gene. This gene is found on plasmids or transposons ie small genetic elements which are capable of moving from one bacterium to another and integrating into the host chromosomal DNA. Copies of the erm gene are transported to other bacteria via plasmids or transposons thru=ogh polite channels. The erm gene is incorporated into the new bacterial genome.
Although selective breeding and radiation therapy already give penicillin a storied history in biotechnology, the advent of new genetic engineering techniques that allow for designer drugs to be produced have kept penicillin on the forefront of antibiotic treatment. Often, the mechanism of action is still the same as far as the chemical structure is concerned. However, the means of production are much more efficient in terms of cost and evaluating new strains of bacteria. These two qualities are highly desirable in the field since bacteria have the ability to quickly mutate and render standard antibiotics ineffective.
Antibiotic resistance is the ability of a microorganism to resist the effects of an antibiotic that would have originally affected the microorganism. The emergence and spread of antibiotic resistance is one of the most pressing world health concerns of the 21st century. In order to combat this spread, the phenomenon must first be understood. Many studies have delved into understanding the emergence of antibiotic resistance, most of which reached the conclusion of Darwinian selection being the reason for the resistance. One of the understood contributions of antibiotic resistance are the toxin-antitoxin systems, which maintain multi-resistant plasmids. In order to combat resistance, researchers have studied cell wall hydrolases, and using them