Pglo Lab Report

a. LB+plasmid and LB-plasmid: Both of these plates had a lawn of bacteria. This proves

The bacteria that was used in the experiment was Escherichia coli which also has the ability to communicate through quorum sensing. Quorum sensing controls bioluminescent, antibiotic production, competence this better understand the bacteria that we were working with (Bassler, 2012). We wanted the bacteria to express antibiotic resistance, bioluminescent and growth all of that is control by quorum sensing which is a type of communication between the populations of the Escherichia coli. In addition, Escherichia coli is a gram negative bacteria which appear as purple when is stained. Gram negative have a thin layer of peptidoglycan but the outer membrane has porins which lets antibiotics such as ampicillin to move through the wall and the bacteria can become resistant to antibiotics. In the experiment, one of the way we made the Escherilia coli was make them competent to allow them the express and share the important information between themselves in the form of genes in the selected plasmid. Plasmids help the bacteria to transfer important date for its survival and its one of the features of the plasmids that is used in the transformation. The Escherilia coli first needed to become competent to take the DNA with the help of calcium chloride

I think that Pencil #2’s (Garlic was placed on specimen before bacteria started to grow) will show fewer bacteria growth than the other specimens.

coli cells, using a green fluorescent protein (GFP), to make them glow. GFP is what causes the bioluminescent jellyfish, Aequarea victoria, to glow in the dark (Cramerie and Kitts 1996). Another piece of valuable information is that E. coli is killed off in the presence of ampicillin. Therefore, we used the plasmid pGLO, which contains the green fluorescent protein as well as an ampicillin resistance. Additionally, the sugar, arabinose, was needed to allow the GFP to glow ( Cramerie and Kitts 1996). I hypothesise that the addition of ampicillin and arabinose will allow us to see how much of the E. coli was transformed by killing off the not transformed E. coli, while enabling the transformed cells to glow. Assuming the transformation is successful, the transformed E. coli will continue to grow and glow, regardless of the presence of ampicillin. The significance of this experiment is that it helps us better understand genetic transformation. Once we have a good grip on the topic, we can then use it to further human medicine. For example, humans that have diabetes cannot produce enough of their own insulin. Instead, they rely on insulin being pumped into their bodies. This insulin used to come from pigs- which was rejected by many people’s bodies. Because of genetic transformation, we are able to produce mass amounts of a person’s own insulin by transferring DNA from them into the insulin of a pig, to create something the human’s body could

Bacterial transformation is the result that occurs when exogenous genetic material is inserted into a prokaryotic cell. The new genotypic makeup translates into a new phenotypic expression. In the case of the lab, the plasmid, pGreen was introduced to the experimental group. In both the control and the experimental group, the existing bacteria was E. Coli MM294. Plasmids are parts of DNA that carry genes. The particular plasmid that was used in this lab was pGreen, which carries the genes GFP and ampR. GFP codes for a protein that makes the bacteria a fluorescent green color and ampR codes for a protein that makes the bacteria resistant to ampicillin, an antibiotic. The fluorescent green color is a trait needed for identification while the ampicillin resistance is crucial for the survival for the bacterial colonies.

coli) with a pGLO plasmid, which will cause it to take in and express the Green Fluorescent Protein (GFP) gene. This gene, which has been encoded into the pGLO plasmid, is found in jellyfish and causes them to produce the protein that makes them fluorescent green. A plasmid is a circular piece of DNA found within a prokaryotic that contains certain genes for a trait that can help the prokaryote to survive. They can also be extracted from prokaryotes and modified to express a desired gene. Bacteria naturally exchange plasmids, causing them to gain traits from the other and increase their chance of survival. To integrate plasmids into DNA, restriction enzymes cut the DNA at certain points, creating “sticky ends”. Sticky ends are areas of unmatched nucleotide bases that can be attached to corresponding nucleotide bases. Once the DNA and plasmid have been cut, the sticky ends from both are attached together so that their nucleotide bases match and are sealed with the enzyme ligase. After this, the cell can express the gene that the plasmid contains. If the E. coli bacteria successfully take in the plasmid containing the GFP gene, they will express the protein and become fluorescent green. The pGLO plasmid also contains a gene called beta-lactamase that causes resistance towards the antibiotic ampicillin. Therefore, if the E. coli bacteria successfully take in the plasmid, they will

Bacterial Transformation is the process by which a foreign plasmid is introduced into bacteria and for the bacteria to amplify the plasmid in order to make large quantities of it. This process has allowed scientists to link genes from different sources of bacterial plasmids, so that the organisms that have been transformed, their cells carry beneficial traits. In this experiment, we used harmless E.coli as the bacteria that was transformed to have the plasmid that had 2 genes: gene for resistance to ampicillin and the gene for the ability to glow green under UV light. The plasmid that we used for the transformation was engineered to contain the DNA sequence of the GFP(green fluorescent protein) gene and the Ampicillin Resistance gene. Also,

The results obtained in the bacterial culture plates were the following: In Plate # I, the bacteria grew as a lawn of bacteria, making it uncountable. In plate # II without pGLO plasmid, there were no bacteria, all of them died in the presence of ampicillin. In plate # II with pGLO plasmid, the bacterial culture lived and grew. In plate # III with pGLO plasmid and araC protein, the bacterial culture grew and glowed. The transformation efficiency is the number of colonies on LB/amp/ara plate divided by the amount of DNA on the plate. Figure 1 shows the culture plates under visible light, figure 2 shows the culture plates under ultraviolet light, table 1 shows the characteristics of the culture plates obtained after the experiment, and Table 2 shows the calculations done for finding the transformation

If genetic transformation allows the insertion of a gene into an organism to gain the trait that the inserted gene codes for, then when 250 µl of transformation solution(CaCl2) and 2-4 colonies of bacteria, E. coli, are added to two micro test tubes, labelled +pGLO and –pGLO, with a loopful of pGLO DNA solution in the +pGLO tube, and are incubated for 10 minutes, then transferred to a 42ºC heat bath for exactly 50 seconds, and then incubated in ice again for 2 minutes for a heat shock, and then have 250 µl of LB nutrient broth, and lastly 100 µl of the solutions spread across the surfaces of 4 LB nutrient agar plates, LB/amp, LB/amp/ara, LB/amp, and LB, with +pGLO, +pGLO, –pGLO, and –pGLO used, respectively, the plates with the –pGLO solution will not glow under a UV light after they have

Genetic transformation occurs when an organism is modified by the introduction of new genetic information which is then incorporated into the organism’s genome. In this lab, engineered proteins containing DNA to absorb blue light and emit green light were incorporated into E.coli bacteria. The main purpose of this lab was to transform E. coli by forcing it to uptake plasmid containing an ampicillin resistance gene and a green fluorescent protein (GFP) using complementation (adding antibiotic resistance in addition to weed out non-transformed cells). To see the effects of this plasmid on cells, bacteria treated with the plasmid were grown 3 different growth mediums: agar, agar/ampicillin(+), agar/ ampicillin(+)/PTG(+). With the plates prepared,

Of the six petri dishes 2 experienced a lawn, two no growth, and two were isolated. Only one plate experienced fluorescence which was the pGLO + on a medium of LB amp + ara. The pGLO- on a medium of LB experienced a lawn of colonies since an LB medium is perfect for growing E. coli coupled with an incubation of 37 degrees. The presence of ampicillin inhibits cell synthesis in E. coli, with that being said the pGLO- plated with LB amp did not grow due to this (Rogers and Mandelstam, 1962). Both the pGLO + plated with LB/amp and LB/amp/ara were able to grow due to the resistance of ampicillin and the 8 microliters of pGLO added to pGLO + tube. In the pGLO + plated with LB/amp/ara the arabinose caused for transcription of GFP to occur resulting

If Genetic transformation has the meaning of “change caused by genes” and involves the placing of a gene into an life form in order to modify the organisms characteristic; the progression of placing genes from one life form to a different is used to assist of a plasmid and the pGLO plasmid codes the gene used for GFP as well as the gene for resistance to ampicillin. It is used to manage the expression of the fluorescent protein; hence, the GFP gene is able to be switched on by adding the sugar arabinose to nutrient medium of the cell, then the bacteria will be able to glow a bright green underneath UV light when arabinose is within the nutrient agar medium. Hence, then when one micro test tube +pGLO and –pGLO are labeled and placed into a foam rack and the tubes are open and using a sterile pipet used to transfer 250 micro liters of transformation solution (CaCl2 ) in each tube, position the two tubes on ice, pick up 2-4 colonies of bacteria with a loop, submerge the loop into the +pGLO tube, repeat steps for –pGLO, put in to ice, and put plasmid DNA into the pGLO; after the pGLO’s need a heat shock by placing the cold tubes into the 42 degrees Celsius hot bath for 50 seconds and back into ice for 2 minutes, later insert the 250 micro liters of LB nutrients broth into the tube and then placing 100 micro liters into the 4 plates, each individual plate contains +pGLO LB/amp, +pGLO LB/amp/ara, -pGLO LB/amp or –pGLO LB). If bacteria that contains +pGLO plasmids is resistant to

This unit had two labs that developed on the same basic idea: gene expression in bacteria. Gene expression is a highly regulated process in which cells either allow or prevent a certain trait to be shown in the next generation of offspring. One major proponent looked at in the first lab was the DNA plasmid. A DNA plasmid is a short, circular sequence of DNA nucleotides that are common in bacteria, but are rarely found in larger organisms. The plasmids are useful in that they introduce new genetic instructions into bacteria cells. In the case of this lab, the new genetic instruction was that of the Green Fluorescent Protein, better known as GFP. This protein was monitored throughout the experiments as the gene that was expressed or prevented. Along with GFP, these plasmids would often pass along a selection gene like beta-lactamase that would allow for the gene to be expressed in specific environments. One example of such an environment is an ampicillin-rich environment.

Genetic Transformation is the process by which a segment of DNA of a specific organism is implanted into another organism that reads the foreign organisms DNA and presents the phenotype for the selected DNA segment.  The genetic transformation can occur using three different methods, but in the case of the experiment conducted and described below, was performed using the method of heat shock. In the experiment, heat shock was used to genetically transform a piece of DNA from a jellyfish into a sample of E. coli bacteria to observe if the transformation would occur. The components necessary for the transformation were: pGLO, a plasmid acting as a vector for the transformation and contained the DNA segment extracted from jellyfish that encodes for the production of a protein that glows (GFP); Luria Bertani broth, the culture for the transformation; Ampicillin Resistant gene, to sort cells so only the cells having absorbed plasmid will survive; Arabinose, a sugar to aid in the growth of bacteria. The genetic transformation was tested using four samples, each having a different environment. The first sample’s environment consisted of +pGLO, the Luria Bertani broth, and a gene resistant to Ampicillin. The second sample’s environment consisted of +pGLO, the Luria Bertani broth, a gene resistant to Ampicillin, and arabinose. The third sample’s environment consisted of -pGLO, the Luria Bertani broth, and a gene resistant to Ampicillin. The fourth sample’s environment

Bacterial transformation is the process of transferring bacteria. This begins with genetic transformation where genes transfer from an organism to another with the help of plasmid. Plasmid contains one or more piece of DNA within bacteria. This technique is used commonly in technology specifically designed for biology usage to enhance the culture around with positive results. It is also used to solve common world problems, such as human insulin and drought resistant crops. In this experiment, the attention was focused on the bacteria, Escherichia coli, also known as E. coli, that is commonly found in intestinal stools. This experiment was conducted in order to test the hypothesis that the GFP will glow underneath the UV light if +pGLO +Ara (Arabinose sugar) were present. It is an overall interesting investigation because bacterial transformation has benefits in agriculture, bioremediation, and even in medicine today that can help with gene therapy.