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
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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
The first step of the experiment was ligation, and the objective was to insert EGFP cDNA into a restriction cut pET41a(+) vector to obtain a recombinant plasmid that would express green fluorescent gene. pET41a(+) was the choice of vector to ligate the EGFP into. Its structural design and genomic sequential properties render it especially well-suited for cloning and high-level expression of peptide sequences. This 5933 bp circular vector contains a built in sequence for Kanamayacin resistance gene. “Rooting of non-transgenic shoots was completely inhibited in all culture media containing kanamycin” (Montserrat, et. al., 2001). This allowed the growth of recombinant and non-recombinant colonies of E. coli, all of which contained the vector insert.
Digestion of the haemolytic and non-haemolytic cells allowed for easier identification of fragments during electrophoresis analysis. Lane 12 in figure 3 show the size markers of SPP1 digested with EcoR1 while lanes 6 and 7 show samples of pK184hlyA and pBluescript digested with EcoR1 and Pst1. Lane 4 was loaded with plasmid DNA from haemolytic cells digested with EcoR1 and Pst1 while lane 5 was loaded with EcoR1 and Pst1 digested DNA from non-haemolytic cells. There was a lack of technical success in both lanes due to no bands appearing in lane 4 and only a single band appearing in lane 5. Theoretically, two bands should appear in both lanes after successful to allow for fragment identification. A possible explanation for the single, large fragment in lane 5 is that successful digestion did not take place and the plasmid was only cut at one restriction site leaving a large linear fragment of plasmid DNA. The absence of bands in lane 4 could be because there was not enough plasmid loaded into the lane. Another possibility could be that low plasmid yield as obtained when eluting the experimental samples in order to purify it. Lanes 8 and 9 belonged to another group and show technical success as two bands were present in both the haemolytic (lane 8) and non-haemolytic (lane 9) lanes. If the
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.
...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.
Therefore colonies containing the non-recombinant pUC19 plasmid have a functional lacz’ gene appear blue on the agar and colonies containing recombinant pUC19 would have a non-functional lacz’ gene due to insertional inactivation and appear white on the growing medium.
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.
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.
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.
Bacterial resistance to antibiotics has presented many problems in our society, including an increased chance of fatality due to infections that could have otherwise been treated with success. Antibiotics are used to treat bacterial infections, but overexposure to these drugs give the bacteria more opportunities to mutate, forming resistant strains. Through natural selection, those few mutated bacteria are able to survive treatments of antibiotics and then pass on their genes to other bacterial cells through lateral gene transfer (Zhaxybayeva, 2011). Once resistance builds in one patient, it is possible for the strain to be transmitted to others through improper hygiene and failure to isolate patients in hospitals.
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.
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.
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