Will transformed E.Coli bacteria be able to undergo mitosis (grow and divide), and glow a green color under a blacklight if it’s placed on a nutrient media plate containing the sugar arabinose and ampicillin? The first step in conducting the experiment was setting up the benchtop following sterile technique. A solution of 30% ethanol and 70% water was used to sterilize the field, and all dishes were put into an autoclave. The next step was putting the bacterial culture onto different nutrient plates using materials from Bio-Rad Laboratories’ pGLO Bacterial Transformation kit. First, 250흻l of the transformation solution was put into two microtest tubes, one labeled +pGLO (the tube administered the plasmid) and the other labeled -pGLO (the tube …show more content…
Coli. The most prominent growth was seen on the plate with Luria-Bertoni (LB) broth and ampicillin (amp) treated with +pGLO, as there were four separate and defined bacterial colonies with surrounding satellite colonies, each of which contained upwards of 20 cells visible to the human eye. Similarly, the plate with LB, amp, and arabinose (ara) treated with +pGLO showed three defined colonies, but no satellite growths. However, when the UV light was used to examine the bacterial colonies of that plate, they gave off a green fluorescence absent from other plates. Comparatively, the LB plate treated with +pGLO showed approximately equal growth to both the LB/amp/ara, and LB/amp plates. However, when examining the -pGLO plates, it was clear that there was conclusively no growth nor was there any fluorescence when the UV light was shown on the cells on either of the plates containing ampicillin or arabinose. However, the LB plate did show immense growth, such that the cells formed a bacterial lawn, yet the cells were not fluorescent under UV …show more content…
Coli can in fact undergo mitosis on plates containing ampicillin, and show fluorescent qualities on a plate containing arabinose. These results logically follows from the fact that the plasmid inserted possesses qualities that allow for ampicillin to be broken down, and therefore not harm the E. Coli, meaning growth on plates containing ampicillin is proof of genetic transformation. Similarly, transformation of the E. Coli is also evident on the plate containing arabinose, as there was not only growth, but clear fluorescence under the blacklight, as the plasmid also codes for that expression. It is clear that those results are a result of the plasmid, as the plates treated with the +pGLO solution can be compared to those with the -pGLO solution, in which there was no growth on any plate except for the LB broth plate. Growth on the LB plate indicates that the E. Coli is healthy, and capable of mitosis in certain conditions, but lack of growth on the other plates points to it still being wild type. Therefore, it is clear that the +pGLO E. Coli have adopted new genes that allow for new functions that wild type E. Coli are incapable of, in addition to showing that the genes were transcripted and translated in S phase of mitosis, as daughter cells possess similar qualities, as shown by their ability to subsequently grow and divide. This In further examining the plates treated with the +pGLO solution,
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.
Streak plate technique was used to isolate pure culture for each bacteria (2). The Gram stain was used to determine Gram reaction and morphology of each bacteria (2) Selective and differential media such as, salt agar, MacConkey agar and blood agar were used for bacterial identification (2). Gelatin deeps were inoculated to detect production of gelatinase (2). Starch Agar plate were inoculated to detect amylase (2). Ocular reticle used to determine bacteria size (2). Motility deeps were inoculated to detect motility on bacteria (2). Thioglycollate broth used to determine oxygen requirements (2). Carbohydrate fermentation
...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.
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].
The tubes were immediately put in an ice until E. coli was added. Then, we added a single colony of bacteria to both tubes and added the plasmid to the tube labeled +pGLO and returned the tubes to the ice bath for ten minutes. Next, four agar plates were prepared: one that had ampicillin (amp) and arabinose (ara), two with just ampicillin, and one with no alterations. After ten minutes, the bacteria were moved to a hot water bath for 50 seconds and then put immediately back on ice for two minutes. After two minutes, 250 microliters of nutrient broth were added to both tubes and the bacteria incubated at room temperature for ten minutes.
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
ScienceCompany. 2010. Bacteria Growing Experiments in Petri Dishes. Available from: http://secure.sciencecompany.com/-W54C659.aspx Accessed on 6th February 2010
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
Next, I tirelessly set up a drip-irrigation system that evenly waters the entire bed out of the tubing, piping, and fittings I picked up with my dad from Home Depot.
+pGLO bacteria on the plate with LB/amp/ara grew and glowed green when the sample was exposed to the sample likely due to the inclusion of arabinose which activated and expressed the GFP gene. The –pGLO bacteria that was ampicillin sensitive failed to grow on the LB/amp plate because it had no resistance to ampicillin which then
As different strains exhibit various growth morphotyes, the combination of different culture media (at least two of them) should be used to achieve optimal recovery rates. This is further increased if transport time is minimized, and if specimens are kept at 4 °C at all times during transit.
E. coli is a gram-negative rod bacteria and a facultative anaerobe. This means that it can live with and without the presence of oxygen (1). Their preferred temperature in which they grow is 37 degrees. This is the equivalent to our body temperature, making our bodies very hospitable for E. coli growth (2). E. coli will not grow spores. (3) However, they do possess acidic polysaccharide capsules. (4) E. coli are motile organisms and they move by means of their flagella (1).
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.
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.
are clinical strains which are courtesy from Hospital Sultanah Nur Zahirah was observed under microscope with 100X magnification. Under 100X magnification motility, shape and size of Bacillus sp. can be observe clearly. Unfortunately our group cannot observe the motility of Bacillus sp. during wet mount technique. This is due to wet mount technique easily to dry very quickly under microscope light in other words it is suitable for short-term observation only because organism will die quickly. However, by using hanging drop technique Bacillus sp. motility can be observe because it allows for longer-time observation and suitable to observe motility of bacterial. Bacillus sp. bacteria use their flagella for a swimming motility and the shape of Bacillus sp. is rod shape. The length of Bacillus sp. observed for wet mount technique is 0.56 μm while for hanging drop technique is 0.06