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1 University of Pittsburgh “Glowing” Bacteria—Transformation of E. coli with pGLOTM Plasmid INTRODUCTION Genetic transformation is a very useful tool to the molecular biologist because it allows genes from one organism to be moved into another type of cell in order to change the traits of that cell. Bacterial transformation is the process by which bacteria take in and use a new piece of DNA. If the new DNA contains genes which the bacteria did not already have, then the bacteria will be “transformed” and will exhibit new traits. The molecular biologist begins by treating the bacteria cells with a solution of calcium chloride in order to make cells that will more readily take up DNA—these are competent cells. The positive charges of the chemical partially neutralizes the negatively charged DNA and plasma membrane of the cell so that they will not repel each other, allowing the DNA to pass through tiny pores in the cell membrane and into the cell where it can be used to make proteins. We will be using a non-pathogenic strain of E. coli bacteria that has already been treated with calcium chloride and is ready for the transformation. How do we know when transformation has occurred? Molecular biologists select for bacteria that were successfully transformed by using the new traits, or phenotype, of bacteria that contain the new DNA. A common selection method is antibiotic resistance. If we begin with bacteria that will die when exposed to antibiotic, and transform with DNA that contains a gene to give antibiotic resistance, then it is likely that the cells that live when grown with the antibiotic have taken up the new DNA. The DNA that we will be using for the transformation is a small circular piece of DNA that contains a gene for ampicillin resistance--the pGLOTM plasmid. Ampicillin (AMP) is an antibiotic similar to penicillin, and the ampicillin resistance gene produces a protein called -lactamase that can destroy the ampicillin and allow the bacteria to keep growing. The pGLOTM plasmid also contains a gene that will make the bacteria glow when exposed to ultra-violet (UV) light by producing a protein called Green Fluorescence Protein (GFP) when the bacteria is given a sugar called arabinose (Ara). The arabinose is used as a “trigger” that switches the GLO gene on; therefore the bacteria will glow only when they are grown with arabinose. 2 We will begin the transformation by adding competent E. coli cells to two tubes; the reaction tube (+DNA) to which we will add the pGLO plasmid, and the control tube (-DNA) which will contain everything except the plasmid. After an incubation on ice, the cells are heat-shocked at 42o C, cooled briefly on ice, supplemented with LB broth, then incubated at 37 o C. After the last incubation, the cells from each tube will be plated on three types of LB agar plates: LB only, LB/AMP, and LB/AMP/Ara. The agar plates will be incubated for 24 hours at 37o C to allow the bacteria to grow. After the bacteria have grown, the plates can be observed under white and UV light, and the results of the transformation recorded. Enjoy the project and keep your mind in full gear as you work! EXPERIMENTAL PROCEDURES Important Notes: Micropipettors are expensive and precisely calibrated instruments! Ask for help before changing micropipettor setting! Never attempt to dial above or below volume limits printed on micropipettor! At the lab bench each group should have: Ice bucket w/ice 2 15-ml push-cap culture tubes Permanent lab marker 100-1000 l micropipettor and blue pipet tips 2-20 l micropipettor and yellow pipet tips 1 vial competent cells (keep on ice) Purple 0.5 ml tube with TE buffer (TE) Amber 0.5 ml tube with pGLO DNA (pGLO) 2 ml LB broth 1. Use permanent marker to label the two (2) sterile 15-ml push-cap culture tubes as follows: PD#-Group# --DNA = No DNA added / Control PD#-Group#+DNA = pGLO transformation w/ plasmid added/ Experimental Chill tubes by placing in ice bucket. Keep competent cells and culture tubes on ice as much as possible! 3 2. Use the 100-1000 l micropipettor and sterile blue tip to add 100l of competent cells to each 15-ml push-cap culture tube. Competent cells are delicate! Pipet slowly to avoid damaging the cells! Pipet the cells directly into the bottom of the tubes, so that a pool of cells forms at the bottom. Avoid splashing the cells up onto the sides of the tube. Place tubes with cells back on ice. DO DNA + Tube FIRST in case aliquot is short. * NOTE: Even though it is always more accurate to chose the smallest micropipettor that includes your desired volume in its’ range, this time you are to use the 100-1000l and not the 20-200l to transfer 100l. The reason is because the competent cells are so fragile that you want to use the bigger tip (not the yellow) with the bigger micropipettor to avoid damage when being transferred. 3. Use the 2-20 l micropipettor with sterile yellow tip to add 10l of TE buffer directly into cells in tube labeled --DNA. Close cap, tap tube with finger to mix. Avoid making bubbles or splashing cell suspension up sides of tube. Return tube to ice. This is the control tube. 4. Use a fresh yellow tip to add 10 l of pGLO DNA directly into cells in tube labeled +DNA. Close cap, tap tube with finger to mix, return tube to ice. This is the transformation tube. 5. Allow both tubes to incubate on ice for at least 15 minutes. (Cells could incubate at this step for up to 45 minutes with no problems; 20 minutes seems to be the optimal time.) 6. While cells are incubating on ice, use permanent marker to label six pre-warmed agar plates. Every group should have two of each of the following plates: 2 LB black line 2 LB/AMP black line + blue line 2 LB/AMP/Ara black line + blue line + orange line Divide plates into two sets of three plates, one of each type, and mark as follows: In small print, label all plates around the circumference of the plate on the bottom (agar-containing side) with your initials (be sure all group members can ID from this), the date, and the agar type. One set of plates should also be labeled with +DNA and the other set with –DNA. Each set of plates should have one of each type: LB, LB/AMP, LB/AMP/Ara. EX: PD#-Group# – 12/11/02 +DNA + (on 1 of each plate type for Experimental) and PD#-Group# – 12/11/02 -DNA+ (on 1 of each plate type for Control) 4 7. Following the 15-20 minute ice incubation, the cells in both tubes need to be heat shocked. Important Notes: Timing of the heat shock is very important! It is critical that cells receive a sharp and distinct shock. Carry ice bucket with cells to water bath (or heat block) set at 42 o C and set timer for 45 seconds before starting heat shock step. 8. Remove both tubes of cells from ice and immediately place into 42 o C water bath (or heat block) and start timing 45 seconds. After 45 seconds, immediately return both tubes to ice. Allow incubation on ice for at least one minute. (Additional incubation on ice is acceptable, but it is better to proceed to the next step.) 9. Use a 100-1000 l micropipettor with a Large/blue tip to add 800 l of LB broth to each tube. Use a fresh tip each time. Gently tap tubes with finger to mix. * If you do not use a fresh tip, you could cross contaminate from Experimental to Control and ruin the experiment!!!!! 10. Allow cells to recover by incubating both tubes at 37oC (heat block, water bath, or incubator) for at least 20 minutes. Additional time at 37oC is acceptable and will lead to higher transformation efficiency. After 37oC incubation, cells will be plated on agar plates that have been pre-warmed at 37oC. Read plating protocol carefully before starting. Important Notes: Plating cells requires the careful use of Bunsen burner and ethanol. Extreme caution must be taken to prevent catching ethanol, hair, clothing, or paper on fire! Always hold spreader downward to prevent ethanol drops from falling onto hand or arm! If beaker of ethanol catches on fire, DO NOT TOUCH BEAKER— IT WILL BECOME EXTREMELY HOT!! Call for help to cover beaker and smother flame. 11. a. Lift lid off of 1 of the three agar plates labeled –DNA; do not place lid down on lab bench. Using a 100-1000 l micropipettor with a sterile large/blue tip, add 250l of cell suspension from tube labeled --DNA onto the agar plate marked --DNA. (The volume plated may be adjusted due to the length of time at 37oC, in step 10. If the 37 o C incubation time was over 1.5 hours, then only plate 50l of cells using a 20-200 l micropipettor with yellow tip.) Do not allow suspension to sit on plate too long before spreading! b. Remove spreader from ethanol and briefly pass through flame until ethanol on spreader catches fire. While holding spreader downward, allow ethanol to burn off away from flame. c. Cool spreader by gently rubbing it on surface of the agar away from cells. After cooling, touch spreader to puddle of cells on agar and gently drag it back and forth several times across surface of plate. Rotate plate a quarter turn and continue spreading; repeat several times. Be careful not to gouge agar. *Failure to COOL SPREADER could lead to Killing Bacteria!! d. Replace plate lid. Return the cell spreader to ethanol without flaming. Repeat step 11 with all agar plates, being careful to match tube and plate labels, and using fresh sterile tip to transfer cells to each plate. * You have two more –DNA plates and ALL 3 +DNA plates to do. Use the +DNA cell suspension when plating the +DNA plates. SEE STEP 12 to HELP! 12. Use matrix below as a checklist as --DNA and +DNA cells are spread on each type of agar plate. Match labels on tubes with plate labels! It will be very difficult to screen for transformants if you plate the cells on the wrong plate! Check off each plate as you go!!!!!!!!!! Plate Type Plate Label --DNA Cells on –DNA plates +DNA Cells on +DNA plates LB black ____ 100l ____ 100l LB/AMP black/ blue ____ 100l l LB/AMP/Ara black/ blue/ orange ____ 100l ____ 100l 5 6 13. Let plates sit for several minutes, agar-side down, to allow suspension to become absorbed into agar. After a few minutes, tape set of six plates together, making sure that the plates are all in the same orientation, i.e. agar-side is facing the same direction. 14. Place plates agar-side up in 37oC incubator, and incubate 12-24 hours. 15. Take time for responsible cleanup: a. Any disposable items that have come in contact with E. coli--culture plates and tubes, micropipettor tips--should be placed into burn box, autoclave bag, or bleach solution. b. Discard liquid cultures and broth into liquid waste container. c. Wipe down lab bench with 10% Lysol solution. d. Wash hands before leaving lab. 16. After plates have incubated for 12-24 hours and colonies appear, examine each plate under white and UV light and record observations in the table. Bacterial growth that completely covers the plate is called a lawn. If distinct colonies are observed but are too numerous to be counted, it can be recorded as TNTC (too numerous to count). If colonies are countable, record number. -DNA +DNA Control Experimental Your Growth: Your Growth: LB Glow? Yes/No Glow? Yes/No (4pts each) LB/AMP (4pts Each) Correct? Yes/No Correct? Yes/No Explain What Growth/Glow should be and WHY: Explain What Growth/Glow should be and WHY: Your Growth: Your Growth: Glow? Yes/No Glow? Yes/No Correct? Yes/No Correct? Yes/No Explain What Growth/Glow should be and WHY: Explain What Growth/Glow should be and WHY: 7 LB/AMP/Ara (4pts Each) Your Growth: Your Growth: Glow? Yes/No Glow? Yes/No Correct? Yes/No Correct? Yes/No Explain What Growth/Glow should be and WHY: Explain What Growth/Glow should be and WHY: ADDITIONAL: If you did NOT get the correct growth, you must PROVIDE an EXPLANATION on the back of this SHEET to EXPLAIN what your ERROR could have been!! You will be graded on this too!!! Name: __________________________ PD: _______ SCORE: / 25 QUESTIONS UNCHECKED COPY TO BE ATTACHMENT 2 FOR EACH GROUP MEMBER IN LAB WRITE-UP! I WILL HAVE YOU MERGE GROUPS AND TURN IN A FINAL ? COPY TO BE GRADED FOR ACCURACY! 1. What type of material is added to the E. coli bacteria in order to introduce new traits in the bacteria? Include the new traits in your answer. (3pts) 8 2. Would adding Green Fluorescent Protein to the cells give the same results as the transformation? Think about the long term and if the offspring will be able to glow!!! Explain in Detail what the difference is. (4pts) 3. Why are the E. coli bacteria treated with calcium chloride for the transformation? Explain what it does and what we call the cells that have been treated with it. (3pts) 4. What is the purpose of the negative controls (the –DNA tube) in the experiment and how did it differ from the experimental groups (the +DNA tube)? (3pts) 5. What allows some E. coli colonies to grow on LB/AMP plates after the transformation? Explain what is going on based on the Central Dogma!! (3pts) 6. Why did the +DNA LB plate show a LAWN and the +DNA LB /AMP show TNTC. To answer this, think about what occurred in some of the bacteria but not all during the experiment!! (3pts) 7. Along with UV light, what else must be present to allow the transformed bacteria to glow and why? (3pts) 9 8. What is the advantage for an organism to be able to turn genes like the pGLO on and off? (3pts) QUESTIONS 1. What type of material is added to the E. coli bacteria in order to introduce new traits in the bacteria? Would adding Green Fluorescent Protein to the cells give the same results? 10 Plasmid DNA with the genes for green fluorescent protein and ampicillin resistance ( lactamase) is added to the bacteria. Adding GFP directly to the bacteria may allow it to glow temporarily, but the offspring would not be able to produce the GFP without the gene, and therefore would not glow. 2. Why are the E. coli bacteria treated with calcium chloride for the transformation? The E. coli bacteria are treated with calcium chloride in order to help neutralize the negative charges on the cell membrane and the DNA so that the DNA can more readily enter into the bacterial cells. 3. What is the purpose of the negative control (the –DNA tube) in the experiment? The negative control allows us to better evaluate the results of the experiment. The cells did not grow with ampicillin before the transformation and did grow with ampicillin after the addition of the plasmid DNA, therefore the DNA must have affected the ability of the bacteria to survive in the presence of the antibiotic. 4. Why do E. coli colonies grow on LB/AMP plates after the transformation? If a sample of transformed bacteria from the LB plate is grown on an LB/AMP plate, will they grow? The bacteria that have been transformed with the plasmid containing the ampicillin resistance gene are able to produce -lactamase, a protein that destroys the ampicillin. A sample of transformed bacteria from the LB plate may or may not grow on the LB/AMP plate, depending on whether or not it has taken up the plasmid DNA. 5. What factors must be present to allow the transformed bacteria to glow? The arabinose sugar as well as ultraviolet light must be present in order for the bacteria to glow. The presence of arabinose allows the gene for the GFP to be turned on, and the protein produced. The GFP uses the energy from the ultraviolet light to emit a longer wavelength of light that is visible. 6. What is the advantage for an organism to be able to turn genes on and off? Producing proteins requires a great deal of energy from the cell, therefore it is more efficient to produce only the proteins which are necessary for the growing conditions of the cells. 11 NAMES: ________________________________________________________________ (If Merging Groups – Put Group ID with Members from Each Group PLUS – If Growth is Different between Groups, Please Indicate This Under Your Growth AND Explain at end) -DNA Control Your Growth: +DNA Experimental Your Growth: Glow? Yes/No Glow? Yes/No Correct? Yes/No Correct? Yes/No Explain What Growth/Glow should be and WHY: Explain What Growth/Glow should be and WHY: LB (4pts each) 12 Your Growth: Your Growth: Glow? Yes/No Glow? Yes/No Correct? Yes/No Correct? Yes/No Explain What Growth/Glow should be and WHY: Explain What Growth/Glow should be and WHY: Your Growth: Your Growth: Glow? Yes/No Glow? Yes/No Correct? Yes/No Correct? Yes/No Explain What Growth/Glow should be and WHY: Explain What Growth/Glow should be and WHY: LB/AMP (4pts Each) LB/AMP/Ara (4pts Each) ADDITIONAL: If you did NOT get the correct growth, you must PROVIDE an EXPLANATION below to EXPLAIN what your ERROR could have been!! You will be graded on this too!!! If a Merged Group, this is only for the Group that did NOT get accurate Growth – Denote Group #/ID. 13