Abstract:
Transformation is the process of uptaking naked DNA by a competent cell. The ability of a cell to go through transformation can be natural or induced. The purpose of this experiment was to verify induced transformation in naturally incompetent E. coli HB101. The pGLO plasmid was the means of transformation in this experiment. The pGLO plasmid contains three core genes: the bla gene encoding for ampicillin resistance, the gfp gene encoding for the green fluorescent protein, and the araC gene which activates the gfp gene in the presence of arabinose. The CaCl2 – heat shock method was used to transform E. coli HB101. After plating the bacteria, the petri dishes containing LB as a nutrient were incubated for 24 hours in a 37°C incubator.
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Although transformation occurred in the pGLO+ plates, the efficiency was not high. Studies have shown how other methods like electroporation and ultrasound have yielded high numbers of transformants as opposed to the CaCl2 – heat shock method. Introduction: Transformation is a process of horizontal gene transfer in bacteria, which consists in the uptake of naked DNA and its genetic information by a cell [11]. The ability of a bacterial cell to uptake free DNA is called competence, and the cells that can go through transformation are called competent cells [9]. Some bacteria can be naturally competent like Legionella pneumophila [14], while others are artificially competent like Escherichia coli, although studies have shown that some E. coli strains are naturally competent [2]. Plasmids are extra-chromosomal, circular, double-stranded DNA molecules that code for genes involved in many aspects of microbiology like virulence and antibiotic resistance. They contain the genetic information that is passed on to competent cells during transformation [13]. The process of transformation is very complex and many proteins are involved for the transfer of DNA from one cell to the other [4]. Many concerns are related to the process of transformation, and this is why transformation is widely studied in laboratories. The main concern regards the transfer of antibiotic resistance genes that can be transported from one cell to the competent cell, making the competent cell resistant to antibiotics [3]. The experiment’s purpose was to transform the E.
coli HB101 strain with the pGLO plasmid. The HB101 strain is a wild strain of E. coli that lacks plasmids and it is useful for the study of bacterial transformation [10]. The pGLO plasmid is a genetically modified plasmid containing the bla gene, the gfp gene, and the arabinose repressor (araC) gene [6]. The bla gene encodes the β-lactamase enzyme, which binds to the β-lactam ring of penicillin and its derivatives providing antibiotic resistance to bacteria. The gfp gene is a gene derived from the jellyfish Aequorea victoria and encodes the green fluorescent protein, a protein that is green under UV light. The araC gene encodes a repressor protein that activates the gfp gene when arabinose is present in the medium used. In the pGLO plasmid the genes that permit arabinose catabolism have been substituted by the gfp gene [1]. The medium used was Lysogeny (or Luria) broth, a nutrient rich medium that permits faster E. coli growth [12]. The E. coli were inoculated in a CaCl2 solution, which aids in the binding of plasmids to the lipopolysaccharides (LPS) membranes of E. coli by attracting the negatively charged backbone of the plasmid and the negatively charged core of the LPS with the Ca2+ ions. A heat shock process was also used to facilitate the uptake of the pGLO into the E. coli cells
[5]. Materials and methods: Two small tubes were assigned and were labeled pGLO+ and pGLO-. The test tubes contained E. coli HB101 inoculated in a CaCl2 solution. Using a micropipette, 8 μL of the pGLO plasmid were place into the pGLO+ tube. The pGLO- served as control. The tubes were then placed in an ice bath for 10 minutes. Meanwhile, 6 petri dishes were labeled. Two dishes were labeled LB and only contained Luria broth; two were labeled LB amp and contained Luria broth and ampicillin; two were labeled LB amp+ara and contained Luria broth, ampicillin, and arabinose. One of each different medium served for the plating of pGLO+ E. coli, the other one was for the pGLO- E. coli. After 10 minutes, the tubes were heat shocked in a 42°C heating block for 50 seconds. The tubes were directly placed in the ice bath again, this time for 2 minutes. Using a micropipette, 250 μL of LB broth were placed in the two test tubes. The tubes were then transferred to a 37°C heat block for 10 minutes. 100 μL of the pGLO+ test tube were transferred to the three respective plates with a micropipette. The same procedure was applied for the pGLO- test tube. A glass spreader was immersed in an ethanol solution and then was placed on the Bunsen burner for a few seconds for sterilization. The glass spreader was consequently used to spread each of the 100 μL on each plate, sterilizing with ethanol and Bunsen burner between the different spreads. Each plate was sealed with a plastic paraffin filmed and then placed in a 37°C incubator for 24 hours [8]. Results: Culture Medium Growth Fluorescence PGLO- LB Yes, lawn No LB amp No No LB amp+ara No No PGLO+ LB Yes, lawn No LB amp Yes, few colonies No LB amp+ara Yes, few colonies Yes Discussion: The results for the pGLO- plates showed that the only growth present was the growth on LB. The LB amp and the LB amp+ara plate showed no growth since the E. coli HB101 strain was not transformed with pGLO and were not resistant to ampicillin. Also, none of the plates presented green fluorescence under UV light because of the absence of the gfp gene. For the pGLO+ there was growth on all three plates showing that transformation occurred. The growth on LB was a lawn type growth, whereas on the LB amp and LB amp+ara plates the growth presented few colonies. The only plate that produced a green fluorescence under UV light was the pGLO+ LB amp+ara because the presence of arabinose in the media activated the gfp gene producing the green fluorescent protein [1]. The results for the pGLO+ suggested that transformation yielded low efficiency levels, as most of the bacteria did not become resistant to ampicillin and did not grow. The E. coli that were transformed, though, presented antibiotic resistance and grew on the plate [7]. Some studies have shown that other methods increase transformation efficiency as opposed to the CaCl2 – heat shock method. One method discovered that E. coli are transformed with high efficiency using electroporation [1,7]. This method consists in applying short but intense electrical fields to the bacteria and results in many transformants. This method would yield a larger number of transformants compared to the CaCl2 method used in this experiment [7]. Other studies have shown that low frequency ultrasounds increase transformation efficiency. This method is less dependent of cell types than electroporation. This method could be the better alternative to induce competence of bacteria of the CaCl2 method [10].
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.
The plasmids in lanes 3,4,8 and 9 have been digested using one restriction enzyme and had been cut at one restriction site, resulting in a linear molecule. Comparing lanes 3 and 4 to
This experiment synthesized luminol (5-Amino-2,3-dihydro-1,4-phthalazinedione) and used the product to observe how chemiluminescence would work. The starting material was 5-nitro-2,3-dihydrophthalazine-1,4-dione, which was, after addition of reaction agents, refluxed and vacuum filtered to retrieve luminol. Using two stock solutions, we missed our precipitated luminol with sodium hydroxide, potassium ferricyanide, and hydrogen peroxide, in their respective solutions, in a dark room, to observe the blue light
...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.
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
a) The way that functional groups affect the reactivity of organic compounds is because of their differences in electronegativity. For example, if a compound is more electronegative, it means that it has a tendency to attract a bonding pair of electrons. So at OH-, the alcohol would be more polar as the oxygen attracts the boiling point, the colour, solubility, etc. this is due to bonding. b) What happens in a nucleophilic substitution reaction is that the nucleophiles attack the carbons of a carbon-halogen bond.
The purpose of this experiment involved synthesis of diphenylmethanol using phenylmagenisum bromide and benzaldehyde, using the method called Grignard reaction. Grignard reactions are an important method for new carbon-carbon bond formation as well as for the synthesis of alcohols.
I would suggest to students performing the nitration to make sure their benzoic acid product is very fine and broken up before reacting it, as it has a tendency to clump together when it dries and thus proves very difficult to react in solution. I would also suggest keeping a very close eye on the temperature when adding the sulfuric/nitric acid mixture dropwise, as the reaction has a tendency to spike in temperature
The “Fast Plant” experiment is an observation of a plants growth over the span of twenty-eight days. The objective is to observe how plants grow and use their resources throughout the span of their life. In our lab we observed the Brassica rapa, a herbaceous plant in the mustard family which has a short cycle which makes it a perfect plant to observe in this experiment. Like other plants the Brassica rapa must use the resources in the environment to create energy to complete itʻs life cycle and reproduce. By observing the plant it is easy to see in what organ or function the plant is using itʻs energy and resources and if overtime the resources switch to other part of the plants. By conducting this experiment we are able to observe where and how plants allocate their resources throughout their life by harvesting plants at different points in their life.
In this work, the mechanical and barrier properties were examined for Polypropylene (PP) film in which the surface of the film was modified by Oxygen plasma treatment. The PP film was treated in various intervals of time of 60 s, 120 s, 180 s, 240 s and 300 s with three various RF power settings of 7.2 W, 10.2 W, 29.6 W. The contact angle was measured to characterize the wettability. The oxygen functional groups were generated on the surface of oxygen modified PP which was observed by Fourier transform infrared spectroscope and it was resulted in the improvement of wettability. The surface morphology and roughness of the PP films before and after the oxygen plasma treatment was analyzed by Atomic Force Microscopy (AFM). It was found that the roughness of
There were two main goals of this investigation. The first one was to find an equation that would produce the perfect number of moves for any number of disks that are being used in the puzzle. The second goal was to find a pattern between all of the different number of disk puzzles. The first goal of finding an equation was accomplished through trial, error, and logical thinking. By first graphing the data points, many equation types were able to be eliminated and a focus was put on exponential equations until the equation that worked perfectly with the data was found. The second goal was also accomplished in a similar manner. When there was not a clear correlation between the 3 disk puzzle and the 4 disk puzzle, the 5 disk puzzle
Bacterial growth may be controlled by many methods; the techniques relevant to this experiment include heat, ultraviolet (UV) light, and antimicrobial control. Using heat as a means of controlling bacterial growth is favorable because it is quick, safe, and cost-effective (Nester, 2007). There are two kinds of heat: moist heat, which destroys the proteins of microorganisms by boiling or steaming, and dry heat, which requires high temperatures to oxidize cell components and damage proteins by incineration or dry heat ovens (Nester, 2007). Cellular proteins are essential in carrying out important biological activities, so without them, the bacteria will not be able to survive (Nester, 2007). Moist heat is widely used to treat drinking water,
Introduction: A phase change is a result from the kinetic energy (heat) either decreasing or increasing to change the state of matter (i.e. water, liquid, or gas.) Thus saying, freezing is the phase change from a liquid to a solid which results from less kinetic energy/heat. Also, melting is the phase change from a solid to a liquid which results from adding kinetic energy/heat. So, the freezing and melting point of something is the temperature at which these phase changes occur. Therefore, a phase change will occur when a vial of 10 mL of water is placed into a cup of crushed ice mixed with four spoonfuls with 5 mL of sodium chloride for 30 minutes. If 10 mL of water is placed in an ice bath, it will then freeze at 5 degrees Celsius because the kinetic energy will leave quicker with the ice involved. The purpose of this lab is to observe what temperature the water must be to undergo a phase change.
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.
The birth of genetic engineering and recombinant DNA began in Stanford University, in the year 1970 (Hein). Biochemistry and medicine researchers were pursuing separate research pathways, yet these pathways converged to form what is now known as biotechnology (Hein). The biochemistry department was, at the time, focusing on an animal virus, and found a method of slicing DNA so cleanly that it would reform and go on to infect other cells. (Hein) The medical department focused on bacteria and developed a microscopic molecular messenger, that could not only carry a foreign “blueprint”, or message, but could also get the bacteria to read and copy the information. (Hein) One concept is needed to understand what happened at Stanford: how a bacterial “factory” turns “on” or “off”. (Hein) When a cell is dividing or producing a protein, it uses promoters (“on switches”) to start the process and terminators (“off switches”) to stop the process. (Hein) To form proteins, promoters and terminators are used to tell where the protein begins and where it ends. (Hein) In 1972 Herbert Boyer, a biochemist, provided Stanford with a bacterial enzyme called Eco R1. (Hein) This enzyme is used by bacteria to defend themselves against bacteriophages, or bacterial viruses. (Hein) The biochemistry department used this enzyme as a “molecular scalpel”, to cut a monkey virus called SV40. (Hein) What the Stanford researchers observed was that, when they did this, the virus reformed at the cleaved site in a circular manner. It later went on to infect other cells as if nothing had happened. (Hein) This proved that EcoR1 could cut the bonding sites on two different DNA strands, which could be combined using the “sticky ends” at the sites. (Hein). The contribution towards genetic engineering from the biochemistry department was the observations of EcoR1’s cleavage of