Elizabeth van Dijk 04 01 14 BIO 183 006B Gene Transformation of the pGLO plasmid to E coli Abstract The purpose of this lab was to transfer the DNA plasmid pGLO to an E coli colony and to become resistant to ampicillin and express the GFP gene Four separate colonies were tested in order to observe the transformation of genes Two colonies had the pGLO introduced but only one was in the presence of arabinose Two other colonies were not introduced to the pGLO but only one was in the presence of ampicillin The results showed that E coli is not naturally resistant to ampicillin and that only in the presence of arabinose will the GFP gene in pGLO be expressed This demonstrates the success of gene transformation using heat shock Introduction This lab was conducted to observe gene transformation specifically pGLO DNA into E coli cells and to successfully use the plasmid to produce ampicillin resistance and a green glow under UV light Gene transformation is the transfer of a gene into an organism in order to change the trait s of that organism Biotechnology consists of gene transformation and is used in agriculture bioremediation and medicine In a similar experiment John Hill and others constructed two yeast E coli shuttle vectors YEp351 and YEp352 and used gene transformation to transfer specific properties into yeast chromosomal DNA In our experiment we attempted to transfer a gene that codes for Green Fluorescent Protein GFP This protein is found in the bioluminescent jellyfish Aequorea victoria It is this particular gene that allows the jellyfish to be bioluminescent in the dark If the experiment is successful we should be able to see this effect in our bacteria under UV light To transfer this gene we must transfer a piece of DNA called a plasmid These smaller circular pieces of DNA usually contain genes for a certain number of traits that could be used beneficially In nature bacteria can complete gene transformation on their own allowing them to share traits and adapt This is what causes the occurrence of bacterial resistance to antibiotics The plasmid used in the experiment is called pGLO This plasmid has the gene for GFP and also a gene for resistance to ampicillin an antibiotic However pGLO also has a gene regulation system that controls the expression of the GFP To turn on the expression the bacteria needs to be in the presence of the sugar arabinose We are able to measure the bacteria that are transformed by the antibiotic plates Only bacteria that have been transformed will be resistant to ampicillin Bacteria without the DNA will not survive with ampicillin Also transformed DNA will appear white on plates without arabinose but will glow green within the presence of arabinose This makes ampicillin a selective marker and arabinose the initiator The purpose of the CaCl2 is the coat and stabilize the charges to make a neutral zone The lipid bilayer has negative charges on the phosphate heads Although the adhesion zone is large enough to fit the plasmid through the DNA also has a negative charge which repels it from the bilayer However calcium ions have positive charges and will coat the DNA and bilayer so that the zone becomes neutral Heat shock is a technique that must also be used in order to complete the gene transformation When the heat is lowered the molecules move slower The rapid rise in temperature creates a temperature imbalance that produces a current for the DNA to travel quickly within the membrane The last part of the lab consists of calculated the transformation efficiency This is used to determine the extent to which the E coli cells have been transformed It is hopeful to transform as many cells as possible The number represents the total number of bacterial cells that expressed the green protein divided by the total amount of DNA used Methods In the beginning of the lab the objective is to transform the DNA in three major steps The first step is to transform the DNA pGLO through the cell membrane by using a transformation solution of CaCl2 First label two separate bullet tubes as DNA and DNA and place them in the foam tube rack In the both tubes transfer 250 L of CaCl2 using a sterile transfer pipette The tubes should then be placed into the ice at the table Using a sterile inoculating loop pick up a single colony of bacteria and immerse the loop in the DNA tube Release the colony by spinning the loop between your fingers Once completed place the tube back in the ice and repeat for the DNA tube Next examine the pGLO DNA solution with the UV lamp and note your observation Then insert a sterile micropipette tip into the plasmid DNA stock tube and withdraw 10 L of plasmid DNA Then mix the plasmid DNA into the cell suspension of the DNA tube Keep the tube on ice The next step is the heat shock Incubate the tubes on ice for 10 minutes while making sure the tubes are completely immerged in the ice During the incubation label four prepared agar plates on the bottom Label a LB amp plate as DNA the LB amp ara plate as DNA a LB amp plate as DNA the LB plate as DNA After the incubation transfer the tubes into the hot bath set at 42 C for exactly 50 seconds After 50 seconds place both tubes immediately back in the ice Keep the tubes on ice for about two minutes and then place them in the foam rack Add 250 L of LB broth to both the tubes using sterile pipettes Incubate the tubes for 30 minutes at 37 C After incubation tap the closed tubes to mix Using sterile pipettes pipette 100 L of the tube substances into the appropriate plates For each plate use a sterile loop to spread the suspensions evenly around the surface of the agar plate Do not pierce the agar The third step is to provide them with the proper nutrients and incubate them for a short period of time to let them recover and begin to express the gene transferred Stack the plates and tape them together Place the stack upside down in the incubator at 37 C for 1 2 days After the incubation observe your results in normal light and ultraviolet light Then investigate the success of gene transformation by calculating the transformation efficiency Results The results of the number of colonies on each plate can be seen in the table below E coli LB amp DNA LB amp ara LB amp DNA LB DNA transformation DNA with pGLO of colonies None 11 None Lawn Amp resistance Yes Yes No No White or green White White White White under normal light White or green White Green White White under UV light GFP expressed No Yes No No The transformation