Science Research paper
The process of genetic transformation is performed by the introduction, uptake and expression of foreign DNA in a new cell. And the path leading to this epoch-making discovery began in 1928 with the work of an English bacteriologist, Fred Griffith. The first demonstration of bacterial transformation was done with Streptococcus pneumoniae and led to the discovery that DNA is the substance of the genes. Moreover, the successful transformation of a cell is usually identified by the expression of a new trait. To do this In experiment for the new trait that is expressed is the pGLO plasmid which has many other different types of pGLO, and we used all those types of pGlo to success in this experiment. And those types are bla gene, GFP gene, araC gene, and ori gene. They all have different their own function in the pGLO plasmid.
Part 1: History of bacterial transformation
Transformation is the process by which foreign DNA is introduced into a cell. Transformation of bacteria with plasmids is important not only for studies in bacteria but also because bacteria are used as the means for both storing and replicating plasmids. Bacterial transformation is the process by which bacterial cells take up foreign DNA molecules from other bacteria, it can then combine it with its own as it replicates or just replicate it. So, if a bacterium takes up a piece of DNA and integrates it in its genome, it is called transformation. Genetic transformation mean “change caused by genes”. Bacterial transformation experiments done by Frederick Griffith (in London) in 1928. Frederick was the first experiments who recommend that bacteria are able of transferring genetic information through a process known as transformation. He use two different types of the bacterium, which infect mice - a type III-S (smooth) and type II-R (rough) strain. Type III-S bacteria was heat killed, its DNA survived and was taken up by the II-R bacteria strain. The Type III-S DNA enabled the II-R bacteria to grow a protective capsule, gain dangerous equity and defeat the host's immune system. Bacterial transformation experiments done by Frederick Griffith (in London) in 1928. Frederick was the first experiments who recommend that bacteria are able of transferring genetic information through a process known as transformation. He use two different types of the bacterium, which infect mice - a type III-S (smooth) and type II-R (rough) strain. Type III-S bacteria was heat killed, its DNA survived and was taken up by the II-R bacteria strain. The Type III-S DNA enabled the II-R bacteria to grow a protective capsule, gain dangerous equity and defeat the host's immune system. In this Activity, I learn about the process of moving genes from one organism to another with the aid of a plasmid. Plasmids are one or more small circular pieces of DNA, which is from one large chromosome. The way plasmids related to transformation is that bacteria can transfer plasmids back and forth allowing them to share these beneficial genes.
Part 2: pGLO transformation
Our bacteria transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E.coli cells and provide an environment for the cells to express their newly acquired genes. To move the pGLO plasmid DNA through the cell membrane, first we use a transformation solution containing CaCl2. Second we carry out a procedure referred to us heat shock. For transformed cells to grow in the presence of ampicillin we must: provide them with nutrients and a short incubation period to begin expressing their newly acquired genes. Moreover, we mixed the plasmid DNA with chilled cells and incubated on ice to allow the plasmid to come into close contact with the cells. The plasmid-cell mixture then is briefly heated the tubes at 42oC for exactly 90 seconds. Return the cells to ice for 1-2 minutes.The heated mixture is then placed back on ice to retain the plasmids inside the bacteria. Many cells do not survive the rapid temperature change but enough maintain integrity to keep the plasmid. After 16 - 20 hours later, we check the plate to see that how many colonies grew on it and count the number of colonies on the plate with well-isolated colonies. For doing our bacteria transformed we used different types of pGLO, such as bla gene, GFP gene, araC gene, and ori gene. They all have different their own purpose in the pGLO plasmid. Such as we use LB agar, ampicillin, arabinose to put the bacteria and the pGLO and T.E buffer solution. We use calcium chloride and CaCl2 to increase the ability of transformation efficiency. We use UV light to examine all components of the experiment and to see the glows and green brightness of GFP.
Part 3: The pGLO plasmid
pGLO is a gene which will make the bacteria glow under UV light. There are different types of pGLO, such as bla gene, GFP gene, araC gene, and ori gene. They all have different their own function in the pGLO plasmid. The +pGLO is the DNA which makes the bacteria glow. The pGLO plasmid is an engineered plasmid used in biotechnology as a vector for creating genetically modified organisms. The pGlo plasmid also contains the arabinose promoter. A promoter is a short region of DNA that regulates the expression of a gene. bla is a gene that gives the bacteria antibiotic resistance. This genes (bla) encoding such resistance can be located on plasmids or the bacterial chromosome. The araC gene which turns on the PGL gene if arabinose sugar is present. The "arabinose regulator," AraC, is a transcription factor that regulates transcription of several genes and operons involved in arabinose catabolism and transport. It coregulated with another transcriptional regulator, CRP; both are transcription factors involved in l-arabinose degradation. These regulators bind cooperatively to activate transcription of five operons related to transport, catabolism, and autoregulation of l-arabinose. Green Fluorescent Protein (GFP) is a protein that glows with a bright green fluorescence on exposure to blue light or UV light. Green Fluorescent Protein is found in the jellyfish Aequorea victoria. The purpose of the GFP gene is to gleam in a dark please to attract. For instance, jellyfish that lives in the deep sea where it is dark has the pGLO gene which glows attract it’s prey. under the protein in jellyfish is that it turns out that GFP is amazingly useful in scientific research, because it allows us to look directly into the inner workings of cells. It is easy to find out where GFP is at any given time: we just have to shine ultraviolet (UV) light, and any GFP will glow bright green. So here is the trick: we attach the GFP to any object that you are interested in watching. For instance, we can attach it to a bactria. Then, as the bacteria spreads through the host, we can watch the spread by following the green glow.
Part 4: pGLO efficiency rate
Transformation efficiency is a number calculated which represents how well the bacteria cells were transformed. How much pGLO we have to add for how many colonies, or the amount of pGLO use for the number of colonies. We calculate this rate by transformation efficiency (transformants/µg) is calculated as follows: # colonies on plate/ng of DNA plated X 1000 ng/µg. The efficiency ratio is important because we don’t want to waste our materials. Also to know the amount of to materials use because we don’t want to use to less or too much. There are some influences that can change the efficiency rate. The most important factors is amount of pGLO. This number useful in real world applications for sensitises, Doctor and other to use for their experiment.
Part 5: pGLO purification
The process of cleaning, removing or cleansing from defilement or uncleanness one molecule to other. Firstable, we already have the mix of bacteria, binding buffer, GFP, Plasmid and Protein and add lysozyme, and freezer. Then, we take the tube and place in the centrifuge to separate the smallest from the largest fro 10 minutes. After that, we have to prepare the chromatography column, the HIC Packaging is going to attract the protein or either repeal thing like water or not. And then, we separate the GFP in one out of the tree tubes. Finally, We separate the GFP from what with don’t want, but there is liquid on it. We have to collect the much liquid and protein as possible. The process purification was used to allow all the liquid buffer to drain from the column. We do that by using the HIC column.
Part 6: Real World application of transformation and purification
One of the main ways is that bacteria are transformed with a human gene and then the human gene is expressed in bacteria to make human proteins for treatment. The protein is purified away from the bacteria and its components before use. Some proteins made in this way include insulin, growth hormone and erythropoietin. Bacterial transformation is used to genetically engineer bacteria to produce medicines. The bacteria responsible for most cases of food poisoning in the U.S. has been turned into an efficient biological factory to make chemicals, medicines and, now, fuels. Chemicals, heavy metals, parasites, fungi, viruses and bacteria can cause food borne illness. Bacteria related food poisoning, but fewer than 20 of the many thousands of different bacteria actually are the culprits. These bacteria are commonly found on many raw foods. Normally a large number of food-poisoning bacteria must be present to cause illness. Therefore, illness can be prevented by controlling the initial number of bacteria present, preventing the small number from growing, destroying the bacteria by proper cooking and avoiding recontamination. Many modern medicines, such as insulin or growth hormones, are made using genetically engineered bacteria. Around this time, when gene transfer became possible, scientists were so fearful that they imposed a voluntary moratorium on this research.
Part 7: The pGLO Efficiency Experiment
Part I
The purpose of this experiment: The purpose of this experiment was to conclude what’s going to be the impact if we more colonies than recommended.
Hypothesis: If we use two colonies of bacteria. Then the number of colonies to transform will increase. Because we used more colonies than recommended before.
Materials: 1)E. coli starter plate ---it is the bacteria we used. 2)LB agar plate, 3)LB/ampillicin plate, 4)LB/ampillicin /arabinose plate,--used to put the bacteria and the pGLO and T.E buffer solution. 5)Sterile microtubes--use to put the liquid mixture. 6)Crushed ice--use for shrink the bacteria. 7)Water Bath--use for open the cell wall to let the plasmid enter it through. 8)20-250 µl micropipet and tips-- use for grab the accurate amount of liquid needed. 9)Inoculation loops--use for to take the plasmid and the bacteria. 10)CaCl2--use for increase the ability of transformation efficiency. 11)LB broth--that’s the food for the bacteria. 12)Incubator --use for heat and hatch the bacteria. 13) pGLO plasmid--use for it’s the plasmid made of bla, araC, ori, GFP gene and ampicillin to glow the bacteria.
Part II
The purpose of CaCl2 is to get the DNA into the bacteria, we have to poke holes in them with the (CaCl2). Also, Calcium Chloride increase the ability of transformation efficiency, which mean is allowing it to cross the cell wall and cell membrane or which can then pass into the cell. The holes poked to allow the DNA in leaves the bacteria leaky. If we don't keep them on ice, they'll 'bleed' to death. We have +pGLO because it has plasmid. And we -pGLO because it don’t have plasmid and it our control. We examine all components of the lab with the UV light at this time to identify several green colonies that are not touching other colonies on the LB/amp/ara plate. And identify several white colonies on the LB/amp plate. Also to note any color differences between the cultures. Incubation on ice because it will not be exposed to the plasmid; therefore, it will NOT inherit the gene. Also, is allows the bacterial membrane to stabilize and to increase the interaction between the calcium cation and the negatively charged components. Is important because if we label the plate and tube we have less chance to make mistake and we are able to know which plate has pGLO and which has pGLO not ampicillin.This step creates pores in the plasma membrane of the bacteria and allows for plasmid DNA to enter the bacterial cell or expand the membrane and allows the plasmid to enter the cells. The reason why we add LB broth to the tubes is because is allows us to create a suspension of bacterial cells from our original colony growing on an agar plate. It provides the necessary nutrients and environment for optimal replication. Is the the food for the bacteria. We use efficiency rate to find how much solution we used for experiment. We start with 250 µl TE buffer with has pGLO and 20ug. Then we divide 20/250=0.08ug/µl.10. We add this to our new tube which already has 250 µl LB, 10µl pGLO, and 250 µl TE buffer and total volume is 510. And then we take 100µl to our agar plate. Then we 100/500= 0.2. Then we 0.8 * 0.2=0.16 µg pGLO. After that we did # colonies/ 0.16ug pGLO. So, we have 33 colonies in our agar plate and we divide 33/0.16 and we 206 and this is our total amount of pGLO DNA (µl) we used for this experiment, which is 206.
Part III
Amount of bacteria on each plate (100ml)
Transformation Procedure
1) Label one closed micro test tube +pGLO. Label tube with your group’s name. Place it in the foam tube rack. 2) Open the tube and, using a sterile transfer pipette, transfer 250 μl of transformation solution (CaCl2) into each tube. 3) Place the tubes on ice. 4) Use a sterile loop to pick up 2 colonies of bacteria from your starter plate. Pick up the +pGLO tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop between your index finger and thumb until the entire colony is dispersed in the transformation solution (with no floating chunks). Place the tube back in the tube rack in the ice. 5) Immerse a new sterile loop into the pGLO plasmid DNA stock tube. Withdraw a loopful. There should be a film of plasmid solution across the ring. This is similar to seeing a soapy film across a ring for blowing soap bubbles. Mix the loopful into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on ice. 6) Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the ice. 7) Label the LB/amp/ara plate: + pGLO. 8) Heat shock. Using the foam rack as a holder, transfer the (+) pGLO tube into the water bath, set at 42oC, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the bottom of the tubes stick out and make contact with the warm water. 9) When the 50 seconds are done, place both tubes back on ice. For the best transformation results, the transfer from the ice (0°C) to 42°C and then back to the ice must be rapid. Incubate tubes on ice for 2 minutes. 10) Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using a new sterile pipet, add 250 μl of LB nutrient broth to the tube and re-close it. 11) Incubate the tubes for 10 minutes at room temperature. 12) Tap the closed tubes with your finger to mix. Using a new sterile pipet for each tube, pipet 100 μl of the transformation in three plates and control suspensions onto the appropriate nutrient agar plates. 13) Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the LB nutrient agar by quickly skating the flat surface of a new sterile loop back and forth across the plate surface. DO NOT PRESS TOO DEEP INTO THE AGAR. 14) Put your group name and class period on the bottom of the stack and place the stack of plates upside down in the 37°C incubator until the next day. 15) Next day , we examine the plates and we found out there were two colonies of bacteria glowing in the UV light.
Discussion
Our hypothesis is if we use two colonies of bacteria. Then the number of colonies to transform will increase. Because we used more colonies than recommended before. And our hypothesis is right because we get average of 53. 3 colonies in 2 plate. This show that more colonies will increase the transform of bacteria.
In conclusion
The planet earth is full of bacteria and they play an important role in the different functions whether they are related to humans, animals or plants. Almost all types of bacteria are helpful to mankind except few species which cause diseases in the human body. And that's the reason why today's scientists are working hard to produce some vitamins which are helpful for the human body. Bacteria are transformed for numerous different reasons. Some of these reasons may include expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease. Other reasons could be expression of proteins that confer on bacteria the ability to survive in particular environments such as to "clean up" contaminated environments in bioremediation. All we have done is good us.
References
Below are the formats for different types of sources:
Web Page with Author
1) Andrew Porterfield. "Bitesize Bio | Who Found the First Plasmid?." 2013. 1 Mar. 2014 <http://bitesizebio.com/13522/who-found-the-first-plasmid/>
2) "Bacterial Transformation and Transfection - The University of ..." 1 Mar. 2014 <http://www.genome.ou.edu/protocol_book/protocol_adxF.html>
3) "Bacterial Transformation." 2008. 1 Mar. 2014 <http://faculty.plattsburgh.edu/donald.slish/transformation.html>
4) "Bacterial Transformation Lab by Matt Love on Prezi." 2013. 1 Mar. 2014 <http://prezi.com/dvdqvb3adsrj/bacterial-transformation-lab/>
5) "Bacterial Transformation Lab." 2010. 1 Mar. 2014 <http://faculty.clintoncc.suny.edu/faculty/michael.gregory/files/bio%20101/bio%20101%20laboratory/bacterial%20transformation/bacteria.htm>
6) "Beneficial and Harmful Bacteria - Biotech Articles." 2010. 1 Mar. 2014 <http://www.biotecharticles.com/Biology-Article/Beneficial-and-Harmful-Bacteria-312.html>
7) "Genetic Transformation of Bacteria with pGLO | Biology." 2012. 1 Mar. 2014 <http://current-biology.blogspot.com/2012/12/genetic-transformation-of-bacteria-with.html>
8) "Genetic Engineering & Transformation - EDVOTEK." 2011. 1 Mar. 2014 <http://www.edvotek.com/Genetic-Engineering-Transformation>
9) "Green Fluorescent Protein (GFP) - Bright Hub." 2009. 1 Mar. 2014 <http://www.brighthub.com/science/genetics/articles/50421.aspx>
10) "Griffith's experiment." 2011. 1 Mar. 2014 <http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Griffith_s_experiment.html>
11) "pGLO™ Bacterial Transformation - Vernier Software & Technology." 2010. 1 Mar. 2014 <http://www2.vernier.com/sample_labs/BIO-A-06A-COMP-pglo_bacterial_transformation.pdf>
These are the question I used those source for;
Bacterial transformation
Who first discovered Bacterial transformation
Who discovered plasmid DNA
How were our bacteria transformed
What was the purpose for LB agar, ampillicin, arabinose, calcium chloride CaCl2, UV light.
What is the purpose of the GFPgene
How are the various techniques and processes used in this exercise used in the world
Calculate the transformation efficiency rate.