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Green Fluorescent Protein II: Bacterial Transformation Objective To get some experience with recombinant DNA techniques and to genetically alter a bacterial strain to produce a particular protein. Background Note that we have not covered this material in lecture yet, so, for today, you will need to do some things without completely understanding them. Even if all the details are not clear, the big picture is based on some pretty simple extensions of the genetics you already know. As the course continues, what you have done today will be clearer. Green Fluorescent Protein (GFP) is a protein produced by the jellyfish Aequoria victoria. The protein fluoresces green when exposed to ultraviolet (UV) light. The goal of today’s lab is to change the genes of a bacterium so that it now makes GFP and will fluoresce in UV light. In today’s lab, you will add a small DNA molecule called a plasmid to a bacterium called E. coli. These bacteria are the workhorse of modern recombinant DNA technology. The bacteria you are starting with cannot produce GFP (since the gene is normally only found in the jellyfish) and are killed by (aka “sensitive to”) the antibiotic ampicillin The plasmid you will be adding is called “pGLO” and it contains two genes: • Ampicillin Resistance – this is a gene that confers the dominant phenotype of resistance to the antibiotic ampicillin. • GFP – this is a gene that produces the GFP protein in bacteria. You will add the pGLO DNA to bacteria that have been treated to make them “competent” – that is, ready to take up DNA from the environment. However, only a very small fraction (fewer than 1 in 1,000,000) of the bacteria will take up the DNA. The bacteria that do take up the pGLO DNA now have the genes on pGLO added to their genome. They have been “transformed”. These bacteria are now resistant to ampicillin and produce GFP. We next select for the transformed bacteria by growing the cells in the presence of ampicillin. Un-transformed cells are sensitive to ampicillin and are killed. However, those that were transformed with pGLO are resistant to ampicillin and will grow. We will be growing our cells on solid medium, so a single surviving cell will give rise to a “colony” of 108 cells – a small pile of cells, all descendants of that original transformed cell. All the cells in the colony will carry pGLO and thus be ampicillin resistant and make GFP. GFP II: Transformation - 3 The genetic map of pGLO is shown below: You can see the genes for ampicillin resistance (AmpR) and GFP; each of these genes has a promoter. We will talk more about plasmids and how they work in lecture. Procedure I: Transformation WARNINGS: 1. In general, the lab is unforgiving of mistakes like using the wrong solution or taking the wrong amount. The construction folks at “This Old House”, say “Measure twice; cut once.“ We’ll adapt this to “Check twice; pipette once”. 2. Although the E. coli strain we use is non-pathogenic (it is not known to cause disease in healthy individuals), you should be careful with it. Always wear gloves, don’t eat or drink in lab, and wash your hands thoroughly when you are all done. 3. Sterile Technique: Contamination is a big problem – it’s a dirty world we live in. You should assume that all surfaces are crawling with nasty microbes. Never let any of the sterile picks, pipettes, etc touch anything except the tube, plate, solution, or colony. If you even think that you’ve touched something you shouldn’t, discard the loop, pipette, etc and get a clean one. GFP II: Transformation - 4 Note that while the figures show plastic transfer pipettes, we will be using pipetmen. Transformation Kit—Quick Guide -pGLO +pGLO 1) Label one closed micro test tube +pGLO and another -pGLO. 1. Label one closed micro test tube Transformation Label both tubes with your group’s name. Place-pGLO. them in the Kit—Quick Guide +pGLO and another 1. Label one closed micro with test tube foam tube rack. Label both tubes your -pGLO +pGLO -pGLO -pGLO +pGLO +pGLO +pGLO +pGLO +pGLO +pGLO +pGLO +pGLO +pGLO -pGLO -pGLO -pGLO -pGLO -pGLO +pGLO -pGLO +pGLO +pGLO +pGLO +pGLO +pGLO QUICK GUIDE GUIDE QUICK GUIDE QUICK QUICK GUIDE +pGLO -pGLO +pGLO -pGLO +pGLO Transformation Kit—Quick Guide +pGLO and another -pGLO. group’s name. Place them in the Label both tubes with your tube rack. 1. group’s Labelfoam one closed micro test name. Place them in tube the +pGLO and another -pGLO. 2) Open the tubes and using P1000 set to “0 2 5”, 250 µl foam tube rack. Transformation Kit—Quick Guide Label both tubes with your transfer 250μl of transformation solution (CaCl2) into 250 µl group’s name. Place them in the each tube. 1. Label foam one closed micro test tube tube rack. +pGLO and another 2. Open the -pGLO. tubes and using a 250 µl Label both tubes with yourpipet, transfer sterile transfer group’s name. Place them in the 2. Open the and using a solution 250tubes µl of transformation Transformation foam sterile tube rack. transfer) pipet, transfer solution (CaC1 into each tube. 2 250 µl of transformation solution Transformation 250 µl 2. (CaC1 Open the tubes and using a solution 2) into each tube. sterile transfer pipet, transfer 250 µl of transformation solution Transformation 3. Place the tubes on crushed ice. solution (CaC1 2) into each tube. 2. Open the tubes and using a Do not use cubed ice. sterile transfer transfer 3. Place thepipet, tubes on crushed ice. 250 µlDo of not transformation solution Transformation use cubed 3) Place the tubes on crushed ice.each Dotube. not ice. use cubed ice. solution (CaC12) into Ice 4. Use a sterile loop to pick up 3. Place the tubes on crushed ice.a single colony of bacteria from your Do not use cubed ice. Ice 4. Use a starter sterile loop pickup upthe a +pGLO plate.toPick single colony of immerse bacteria from yourinto the tube and the loop plate.on Pick up the +pGLO 3. Placestarter the tubes crushed ice.at the solution Ice 4. tube Use and atransformation sterile loop to pick upinto a the immerse the loopSpin bottom of the tube. the loop Do not use cubed ice. single colony of bacteria from your transformation solution at the your index finger and starterbetween plate. Pick up thethe +pGLO bottom of the until tube. Spin loop is thumb the entire colony tube andyour immerse the loopand into the index finger Ice 4. Use abetween sterile loop to pick up a dispersed in the transformation transformation solution at theis thumb until the entire colony (withfrom no floating single colonysolution of bacteria your chunks). bottom of in thethe tube. Spin the loop dispersed transformation 4) Use a sterile loop to starter pickup a Place single colony thethe tube backof in the tube plate. Pick up +pGLO between your index finger and solution (with no floating rack inthe the ice. Using a new sterile tube plate. and immerse loop intochunks). the bacteria from your starter Pick up the thumb until the entire colony is Place the tube back inthe the-pGLO tube loop, repeat for transformation solution at the +pGLO tube and immerse rack the loop thea new steriletube. dispersed ininto the transformation in the ice. Using bottom of5.theExamine tube. Spin the loopplasmid DNA -pGLO the pGLO solution (with floating chunks). transformation solution at loop, the bottom of the repeat forno the -pGLO tube. between your index finger and solution with the UV lamp. Note Place the tube back in the tube tube. Spin the loop between your index finger thumb until the the entire colony is Immerse -pGLO 5. Examine pGLO plasmid DNA rack inyour the observations. ice. Using a new sterilea new and thumb until the entire colony is dispersed dispersed in the transformation solution with loop the UV lamp. Note sterile into-pGLO the plasmid loop, repeat for the tube. DNA solution (with no floating chunks). a loopful. in the transformation solution (with no floating your observations. Immerse new stock tube. Withdraw -pGLO 5. sterile Examine the pGLO plasmid DNA Place the tube back in the tube loop into the plasmid DNA chunks). Place the tube backsolution in the tube rack There should be ain film of plasmid with the UV lamp. Note rack instock the ice. Using a newathe sterile tube. Withdraw loopful. solution across ring. This is the ice. Using a new sterile loop, repeat for the - a new your observations. Immerse loop, repeat for the to -pGLO tube. There should be a film of plasmid similar seeing a soapy film pGLO tube. sterile loop into the the plasmid solution across ThisDNA is soap -pGLO aplasmid ringring. for DNA blowing 5. Examine theacross pGLO stock tube. Withdraw a loopful. similar to a soapy film into the cell Mix the loopful solution withbubbles. theseeing UV lamp. Note There should beblowing a film ofsoap plasmid across a ring Immerse for suspension of the tube. your observations. a +pGLO new solution across the ring. This iscell Mix the loopful intoµlthe Optionally, pipet 10 of pGLO sterilebubbles. loop into the plasmid DNA similar to seeing a+pGLO soapy film plasmid DNA of the tube.tube and plasmid into the +pGLO stock suspension tube. Withdraw a loopful. -pGLO across a ring for10 blowing soap Optionally, pipet µl of pGLO -pGLO tube and return There shouldmix. beClose a filmthe of plasmid plasmid DNA bubbles.into Mixthe the+pGLO loopful tube into the cell plasmid and -pGLO it tothe thering. rack This on ice. add solution across is Do not suspension of the +pGLO tube. Close the -pGLO tube returntube. plasmid DNA film to theand -pGLO similarmix. to seeing a soapy Optionally, pipet 10Do µl of pGLO theWhy rack on ice. not not? Close the add -pGLGO tube plasmid DNA acrossit to a ring for blowing soap plasmidDNA into the +pGLO tube and -pGLO plasmid to the -pGLO tube. to the on ice. bubbles. Mixand thereturn loopfulitinto therack cell mix. Close the -pGLO tube and return Why not? Close the -pGLGO tube suspension of the +pGLO tube. on ice for Incubate the tubes it to6.return the rack onthe ice.rack Do on notice. add and it10to Optionally, pipet µl of pGLO 10 min. Make sure to push theplasmid DNA plasmid DNA to the -pGLO tube. Rack Ice plasmid intotubes thethe +pGLO tube and 6. Incubate tubes on ice for tube -pGLO all the down in the Why not? Close theway -pGLGO mix. Close therack -pGLO tubebottom and return 10 min. Make sure to push the so the of the tubes and return it to the rack on ice. Rack Ice it to the rackall onthe ice. Do not addin contact tubes way down the stick out and make with 18 GFP II: Transformation 6. Incubate tubes on icetubes for - 5 plasmid DNA to the -pGLO tube. rack sothe thethe bottom of the ice. 10 min. sure contact to push the Why not? Close themake -pGLGO tubewith stick out Make and 18 Rack Ice tubes all the way down in the and return it to the rack on ice. the ice. rack so the bottom of the tubes 6. Incubate the tubes on ice for stick out and make contact with 18 10 min. Make sure to push the the ice. -pGLO -pGLO +pGLO +pGLO -pGLO O O O O O O O O +pGLO QUICK GUIDE 8. Heat shock. Using the foam rack 7. While the tubes are sitting 8. on Heatthe shock. Using the foam rack 8) Heat shock. Using rack as aboth holder, as foam a holder, transfer the (+) ice,transfer label yourboth four agar pGLO pGLO as pGLO a holder, transfer both the (+) pGLO pGLO pGLOand and (-) pGLO tubes into theplates (+) (-) pGLO tubes LB/ LB/amp ar a B / a m ptubes into / on the bottom (not the lid) as a m p pGLOthe andwater (-)LpGLO LB bath, set at 42°C, for intoonthe bath, at bath, 42°C, for exactly 50 shown thewater diagram. theset water at 42°C, for sure exactly 50set seconds. Make to seconds. Make sure to push the tubes allway the exactly 50 seconds. Make sure to down push the tubes all the way down in the rack the bottom of the push so the tubes all the way down in the rack so the bottom oftubes the 8. Heat shock. Using foaminrack Water bath the tubes rack so the out bottom of the contact stick out andthemake contact with the warm stick and make Water bath as a holder, transfer both the (+) with tubes stick out and make contact the warm water. When the water. When the 50 seconds have passed, place pGLO and (-) pGLO tubes into with the warm water. When the place 42°C for 50 sec Ice Ice 50 seconds have passed, For the best 42°C for 50 sec theboth water tubes bath, setback at 42°C,on for50ice. Ice Ice seconds haveback passed, place both tubes on ice. For the transformation thetransformation change from the the ice exactly 50 seconds. Makeresults, sure to tubes both back on ice. For the best results, push the tubes all theand way down (0°C) to 42°C then back to thethe ice best transformation results, the tobe rapid. Incubate tubes on ice for 2 min. change from icemust (0°C) in the rack so the bottom of change the 42°C fromand thethen ice (0°C) back to the ice Water bath tubes stick out and make contact 42°C and then the ice tubes must be back rapid.toIncubate with the warm water. Whenmust the on be ice rapid. tubes for 2Incubate min.tubes 250 µl Remove the rack containing the from the 42°C for 50 ice sec and place Iceon the bench top. Open the 50 9) seconds have passed, place on ice for 2 min. Ice 250 µl both tubes back on ice. For using the9. Remove rack containing the 250μl of LB nutrient broth to the tube and pGLO+ tube and, a newthe sterile pipet, add 9. Remove the rack containing the best transformation results, the tubes from the ice and place on pGLO- tube. Incubate the tubes for 30 min at reclose it. Repeat with a new sterile pipet for the frombench the ice and place on and, change from the ice (0°C) totubes the top. Open a tube room temperature.the bench top. Open a tube and, 42°C and then back to the ice using a new sterile pipet, add using 250 a new sterile pipet, add must be rapid. Incubate tubes µl of LB nutrient broth to the 250 µltube of LB nutrient broth to 250 the µlwith on ice for 2 min. and reclose it. Repeat tube and reclose it. Repeat with a new sterile pipet for the other LB-Broth 9. Remove the rack containing the tube. a new sterile pipet for other Incubate thethe tubes for LB-Broth tubes from the ice and place on Incubate tube. tubes for 10 min atthe room temperature. the bench top. Open a tube10 and, min at room temperature. using a new sterile pipet, add10. Gently flick the closed tubes with 100 µl 250 µl of LB nutrient broth the your 10. to Gently flickfinger the closed tubes with to mix. Using a new 100 µl tube and reclose it. Repeatyour with finger topipet mix. Using a new sterile for each tube, a new sterile pipet for the sterile other pipet pipet forµleach tube, 100 fromLB-Broth each of the tube. Incubate the tubes forpipet 100 tubes theeach corresponding µl to from of the 10 min at room temperature. as shown on the diagram tubes plates, to the corresponding the appropriate plates. plates,onto as shown on diagram GFP II:the Transformation -6 10. Gently flick the closed tubes with +p -p -p +p onto the appropriate plates. GL 100 µl GL GL GL your finger to mix. Using a new + p L B / a m p + p LB/am LB/ a- p p r p LB a m a / p L G L L G L G G sterile pipet for each tube,11. Use a new sterile loop for each LB LB/ LB/ /amp/ara LB amp amp pipet 100 µl from each11. of the Spreadloop the suspensions Use aplate. new sterile for each tubes to the correspondingplate. evenly the surface of the Spreadaround the suspensions QUICK GUIDE +pGLO +pGLO +pGLO loop, repeat the tube -pGLO tube. Place the tube backfor in the rack5. in the ice. Using a newplasmid sterile DNA -pGLO Examine the pGLO loop, repeat for the solution with-pGLO the UVtube. lamp. Note your Immerse -pGLO 5. Examine theobservations. pGLO plasmid DNA a new sterile loop into the plasmid solution with the UV lamp. Note DNA 5) Examine the pGLOyour plasmid DNA solution stock tube. Withdraw loopful. observations. Immerse aanew There should be a film of plasmid with the UV lamp. Note observations. sterileyour loop into the plasmid DNA solution across ring. This is Withdraw a the loopful. Using a pipettor, put stock 10μltube. of pGLO plasmid similar to seeing a soapy film shouldClose be a filmthe of plasmid into the +pGLO tube There and mix. +pGLO across a ring for blowing soap solution across the ring. This is tube and return it to the rack on ice. Do not add bubbles. the loopful similar to seeing Mix a soapy film into the cell plasmid DNA to the -pGLO tube. Why not? of the soap +pGLO tube. across asuspension ring for blowing Optionally, pipet 10 µl of pGLO Close the -pGLGO tube and return it tointo the rack bubbles. Mix the loopful the cell plasmid DNA plasmid into the +pGLO tube and -pGLO suspension of the +pGLO tube. on ice. mix.pipet Close10 theµl-pGLO tube and return Optionally, of pGLO plasmid DNA itinto to the on ice. Doand not add therack +pGLO tube -pGLO 6) Incubate the tubes plasmid on ice for 10 min. Make plasmid DNA to the -pGLO tube. mix. Close the -pGLO tube and return sure to push the tubesit toallthethe down the tube Why not? Close thein -pGLGO rackway on ice. Do not add and return toout the and rack on ice. contact rack so the bottom ofplasmid the tubes stick make DNA to theit -pGLO tube. Incubate the-pGLGO tubes ontube ice for Why6.not? Close the with the ice. 10 min. Make to push the and return it to the racksure on ice. Rack Ice tubes all theon way 6. Incubate the tubes icedown for in the rack so the bottom of the tubes 10 min. Make sure to push the stick make contact with Rack Ice 18 tubes all the out wayand down in the the ice. rack so the bottom of the tubes stick out and make contact with 18 theWhile ice. the tubes are sitting on 7. 7. While the label tubes are sitting on plates 7) While the tubes are sitting on ice, label ice, your four agar pGLO pGLO pGLO pGLO LB/ ice, label your four agar plates pGLO pGLO LB/amp onthe the bottom (not the lid) as amp/ara pGLO LB/amp pGLO L B your four agar plates on bottom (not LB/ LB/amp LB/amp on theshown bottomon (not lid) as amp/ara LB thethe diagram. the lid) as shown on the diagram. shown on the diagram. -pGLO +pGLO +pGLO -pGLO +pGLO -pGLO QUICK GUIDE QUICK GUIDE 8. Heat shock. Using the foam rack both tubes ice.set For the back wateron bath, atthe 42°C, for a holder, transfer both the (+) best transformation results, Make the as exactly 50 seconds. sure to pGLO and (-) pGLO tubes into change push from the the tubes ice (0°C) to all the way down water bath, set at 42°C, for 42°C and thenrack back thebottom ice the in the sotothe of the exactly 50 seconds. Make sure to Water bath must betubes rapid. Incubate tubes stick out and make contact push the tubes all the way on ice flick for 2 min. µl 10) Gently thewarm closed tubes todown mix. Using a new sterile pipet for each with the water. Whenwith the your finger 250 in the rack so the bottom of the 42°C for 50 sec Ice Ice on the diagram 50 seconds have passed, place tube, 100ul from each of toand themake corresponding plates, as shown Water bath 9. pipet Remove the rack containing thethe tubestubes stick out contact both tubes back on ice. For the onto the appropriate plates. sure putwater. the cells tubes from the ice and place Be on with theto warm Whenon the the jello-like agar medium, not the besttop. transformation 42°C for 50 sec bench Open a tuberesults, and,50the Ice Ice seconds have passed, place plasticthe lid! change from the ice (0°C) to using a new sterile pipet, add both tubes back on ice. For the 42°C and thenbroth backtotothe the ice 250 µl of LB nutrient best transformation results, the must be rapid. Incubate tubes tube and reclose it. Repeat with change from the ice (0°C) to on ice for 2 min. a new sterile pipet for the other42°C and then back to the ice 250 µl LB-Broth tube.9.Incubate mustthe be rapid. Incubate tubes Removethe thetubes rack for containing 10 min at room temperature. on ice 250 µl tubes from the ice and place onfor 2 min. the bench top. Open a tube and, 9. Remove the rack containing the 10. Gently flick the closed tubes with using a new sterile pipet, add 100 µl place on tubes from the ice and your finger to mix. Using a new 250 µl of LB nutrient broth to the the bench top. Open a tube and, sterile pipet for reclose each tube, tube and it. Repeat with using a new sterile pipet, add pipet 100 µl from each of the a new sterile pipet for the other LB-Broth 250 µl of LB nutrient broth to the tubes totube. the corresponding Incubate the tubes for tube and reclose it. Repeat with plates, as shown on the diagram 10 min at room temperature. a new sterile pipet for the other onto the appropriate plates. LB-Broth + p tubes for + p -p -p tube. Incubate the GL 10. Gently flick the closed tubes with L GL L G G LB/ LB/ 10 min at room temperature. p 100 µl LB/amp/ara p LB m a m a your finger to mix. Using a new 11. Use a new sterile loop for each sterile pipet for each tube, plate. Spread the suspensions 10. Gently flick the closed tubes with pipet 100 µl from each of the 100 µl evenly around the surface of the your finger to mix. Using a new tubes to the corresponding agar by quickly skating the flat sterile pipet for each tube, plates, as shown on the diagram surface of a new sterile loop pipet 100 µl from each of the onto the appropriate plates. back and forth across the plate tubes to the corresponding +p -p -p +p GL GL GL GL surface. plates, as shown on theLdiagram LB LB/ B/amp p /amp/ara LB am ontoeach the appropriate plates. 11) Use a11. new loop loop for each plate. Usesterile a new sterile for +p -p +p GL plate. theevenly suspensions 12. Stack yourSpread plates and tape around the GL GL Spread the up suspensions LB LB/ LB/ a p r / m a amp/a amp evenly around the surface of the them together. Put your group surface of the agar by quickly skating the 11. Use a new sterile loop for each agar by period quicklyon skating name and class the the flat plate. Spread flat surface of a new loop back andthe suspensions surface of asterile newplace sterile loop bottom of the stack and the evenly around the surface of the forth across the plate surface. back and forth the plate stack upside down in across the 37°C agar by quickly skating the flat surface. incubator until the next day. surface of a new sterile loop back and forth across the plate 12. Stack up your plates andsurface. tape them together. Put your group 19 class period the them together. Put your group name and 12) Stack upname yourand plates and12.on tape Stackthe up your plates and tape bottom of the stack and place class periodstack on the bottom of the stack andPut place stack upside down in the them together. your the group upside down in the 37°C name and class period on the 37°C incubator untiluntil thethenext incubator next day. day. bottom of the stack and place the stack upside down in the 37°C incubator until the next day. O O O O O GFP II: Transformation - 7 O O O O Lab Report There is no lab report for this session. O O 19 Once your cells have grown, your TA will put them in the refrigerator so you can look at them during the last week of lab. 19 -p GL LB IGV Practice Many students find it challenging to use the Integrative Genome Viewer (IGV) on the SPOC. In this part of the lab, your TA will help you to learn how to use the IGV by working through some of the SPOC IGV problems. GFP II: Transformation - 8