Background & STUDENT GUIDE [ Date Name $$ Key Question ow can wef buman roti products produced nie aother ei nd why is that important? answer the Key Question. ‘ae bass of what you eared in the introductory Case Stay asi Background sranjersing DNA fom one specs into another species has many PSN applications and has led to great seproverent in buran beth inactive shor time procedure has become # major process? ip pace word The fist success atemps at mering ato a bacterial plasmid Bites 197- In 1976 the nen gene was nse ia « OS and in 1979 the gene for human {fons hrmane wa inserted io abate, These a0 uch led to the large scale production of human oct were pees expense and more elie Wes PE treatments Since that time, dozens of rod et acs he ben engnere sig bacteial wansformation ab wil help you understand how a maar enlnered bacterial Pal can bs mses imei 0% pcteril cl eading tothe potential for harvesting the protein product “hn guide provides bri history of rasformain, dicuon of Mess of transformation, and procedure for ‘conducting the transformation. Discovery of Transformation vy toan the gl sci Frederick oii was staying the bse SPT pneumoniae, the major 198 te Epona, Alta ne, pita was heeding SS fhe Westera Hemisphere carat get tone si Speman oe was paihogls(et isease) andthe other was 0% Se we sn pode a plysccarde coating hat coed 1S ‘on aga appear smooth ee aeapaogene tain di not proce the coating ands core “peste rough (R), We now know that oars te smooth ran pathogenic llowng to ars PME Filled by the hosts immane system. wr a epeiments ave injcting ce wah he wo sis, When ie 88 smooth strain the mice care Tad led. When be used the rough trai hey taped ASST. series of experiments, Grifith bec led non cls with ving rough els Nether ofthese ol ‘ness inthe mice ifused ‘Garey But when sed together in he same mice temic DST ‘and ied jst as if they had been injected Ser math cells Grif was surprise! When he soit = pneumoniae cells rom the dead mice, he wh ae pow produced smooth colonise dss podacng S70 He concluded that, somehow, sea ough els ha ben transformed in smooth celwhen nixed with the dead smooth cel the ving ro epoter ro of estos Aves MCs and Mae 6) Showed thatthe “wansforing Primi he sabtance rom he eat ile smooth stan ta hosed the transformation was DNA. ae s4 cn en sg SeLiving S-cats Living Freetis eo oe eet Et Living & and Feo Gritth's Transformation Experiment ‘Natural Transformation “Pneratonocurs naturally n any ba nol ace Some Pe take up DNA in the environment ra ee ino tr cn DNA. here cough slaty 8 Tse sequences ina homologous 80%. snd ncnper repr theo DNA, Tis rzambnation son ofS 9" hot bacteria species can become ree ny ments versity aos bacteria to eta oles example in Nelseria gonorrhea he sao a hat eases onorhea tansformation cables he oTENA ‘pode the immune system oft ham bacteria at Nara have thn protons made of poten 8 TE their surface. Our immune mete pote anodes othe pin re ows Saslse A Pa einfection by N. gonortheae, st we Sr porn contin several versions fhe pln gee fo ore AS ransformation by DNA containing arn rte pin gene, Ngoorent changes the version of lin ROD it aynhesizes, evading fecognition bythe immune syste antibodies. . aoe ona attra nba cln can bean op yng st ot ee A agent mares wth he DNA. he IVS Om °. Tn i atu tbe agent me denial co cnr ee SCE S2 ‘Bacteria! TransformationsBackground (continued) /srupenr cue [Artificial Transformation Tre sameat ae for aceriat tke up DNA fem te ronment Pats ee bacteria can be meted to codons that enable them take up new DNA mos ca Cell that are in 2 receptive state oF ee DN are rettd tas competent One way makecls comes change the inte strength of kin ON toe the clin the presence of Positive ons asa ce) TEE treatment renders the Ihe pene to DNA Hh volge aslo bse wed to ender oo permeable to DNA, ina process call mere Pee Once DNA bas ern taken pb bates the new DNA GO “expressed” meaning called unened and tratlated to produce protein prdiets mach a 9s SE DNA does In natural aa ae ge DNA ta is incoported int a aces is ery sii Os BE DNA, already found ano alanine DNA 0 be inserted may be wry ten the bacterial genome TO ia tosh foreign DNA is taken up ad expressed by the bacteria sient plasmids 1 plas a relate shot rela pce of dble-sranded DNA nt bacterial and yeast cells. plasmid A pa rpcaton an can reprodce separa rom the aces eh ‘Asa esl, Dacteriam may basi ow casing land Pass havea small sumer ons 298 ‘pica are not necessary canny areal ofthe acter however they do contain gees. EN expressed can confer savial fo Yr ample many asic genes thst make the cele Sa antibties or that eta gt unusal materia, An inroads no ncorporated into a gEnOE a ced a bacterial cel takes plasm the plasmid eres expresed regardless of aac the pl incorporated int the act gnome oF remains SCT the eytoplast, Plasmids are whet podeton of uman product foto main ewsons 1) acer 7 naturally take in sian 2) ities to ncorporatinto 2 plasmid portions of DSA SEAS Samir to a bacteria senomic DNA. puman eat Ste ain plaemid ficial wanstrmetion process fr bacteria production of human ns Bacterial Transformations 33 centr ge 40 oeSO Se peNT GUIDE 9, STUDENT GUIDE sn 197, Pau Regist ed etiam ene nd DNA igs combing froma monkey virus with tee pei vr called Iba ate, Stanley Cen and Heche Boyt ahleto pat recombinant Ori cll Using cession enzyme toy sre agen sees to kanamycin (an OA re outer plastid. Coben had alady developed a method bacteria to take in plasmids, sete ee able reste acral all tht ws esta ana find that passed the resistance Sere, pgny Th texlog of arte betel wensforaton Dal ENE ‘Dozens of products are now produced by means ofthis technology. Selecting for Transformed Bacteria cscs ave designe a asi with the DNA sequenes fineness 601) inserted they prepare soltion Os ge abe of beeps They ct psi sn os of bacteria, hoping that a nea wl ke up he sm La most cases apposite P07) Ton bscteria actully take ‘ork nm: Because cells tat ake up the lami se extra rezone ST the genes in the plasmid, they ina amet wih the non-sansormed aceia, whose grow tends 9 TSE the growth of the a roced bactera, Biotecnologie mest pve te ransformed Base vantage and somehow isolate them tein To make avatar eto retain las nd 0 for use ofa selectable fe inde which bacteria ave been ransormed, chicas ws 97ST" volving antbiotilaced row medium ad genes for esstanceo ano biotechnology. In ation Fe ruanc finest the plosmid aso scnes an anion reins Eo he bacteria ae allowed to ow and reproduce onan aga late ised wit the er ihote only those actriathat ave een transformed we tne anbaticreistance pee a sce mare wid He) transformed bacteria Tete si rw Tat gen a be id ons mir change the sor ofthe colo 0 that they can be “Teangushed om bacteria hat have taken wy the Pasi centr eng bo sa ‘Bacterial Transformations:Laboratory Procedure (continued) Name f ‘STUDENT GUIDE Date Laboratory Procedure for pGREEN 1. Mark one sterile 15-mL tube “+ plasmid” ‘Mark another *-plasmid” (Plasmid DNA will beaded to the "yplasmid” tube; none willbe added tothe “plasmid” tube) Use a sterile transfer pipet to add 250 pL oicecold calcium chloride to each tube, 1,000 ut. (1.00 mt) —>] 780 pt (0.7m —+} 500 pL (0.50 mu) —»| 250 pl (0.25 mt) —> 100 pt (0.10 my) —>| 3, Place both tubes on ie 4 Use sterile plastic inoculating loop to transfer ‘one of more isolated colonies of E co from the starter plate tothe splasmid tube. The total amount picked should equal 2-5 mm in diameter Be careful not to transfer any aga fom the plate alongwith the cell mass, b, Immerse the calls onthe loop in the ‘calcium chloride solution in the +plasmid tube and vigorously spin the loop in the solution to dislodge the cell mass, Hold the tobe up to the ight to observe thatthe ‘ell mass has fallen off the loop, 5. Immediately suspend the cellsby repeatedly Piettng in and outwith asteie transfer pipet. Piet caeflly to avoid creating bubbles ‘or splashing the suspension up the sides ofthe tube. Examine the tube against light to confirm ‘hat no visible chumps of cells remain inthe tube or ar losin the bulb of the transfer pipet. "The suspension should spear milky white ‘Bacterial Transformations: SOAs, erage, reusgend esa a8 roy aoa scenes Vv oe 1-2 minutes eis 250 ‘ad 250 tse st Room temperate 6-15 minutos s2tae STUDENT GUIDE, Laboratory Procedure (continued) 6. Return the splasmid tube to ie. Transfer a mas of cells tothe plasmid tube and suspend as described in steps and 5 above 7, Return the plasmid tube to ce, Both tubes should now be one. 1, Uses sterile plastic inoculating lop to add one loop of plasmid DNA tothe +plasmid tube. (When the DNA, Solution forms a bubble across the lop opening, is volume i 10 ul.) Immerse the lopful of plasmid DNA Aiecly into the ell suspension and spin the logpto mix the DNA with the cells {9 Retur the «plasmid tube to ice and incubate both tubes on ice for 15 minutes. 10, While the tubes are incubating, label your media plates as follows and with your lab group name and date ‘4. Label one LB/Amp plate “plasmid” This isan experimental plate, bi Label the other L8/Amp plate “plasmid” This is a negative control, ‘Label your LB plate either “plasmid” or "plasmid? according to your teacher’ instructions. This {4 positive contol totes the viability ofthe cll after they have gone through the transformation procedure 1, Following the 15-minute incubation on ic, "heat shock" the cells, Carefully carry your beaker of ie and submerged tubes ove tothe waterbath. Remove both tubes directly from ie and immediately immerse them Inthe 42°C waterbath for 90 seconds. Gently agitate the tubes while they are in the wate bath. Return both the tubes dretlyto ce for 1 or more mints. 12, Usea sterile transfer pipet to add 250 ul. room-temperature Luria broth (LB) to each tube. Gently tap the tubes vith your finger to mix the L with the cel suspension, Place the tube in atst tube rack t room temperture fora 5-to15-minate recovery 13, Now you will ramove some cells rom each transformation tube and spread them on the plates Cells from the plasmid tube shouldbe spread on the -plsmid plates and eel from the + plasmid tube shouldbe spread on the plasm plates, 14, Use a sterile transfer pipet o add 100g of cells fom the plasmid transformation tube to each appropriate plate, Using the procedare below; immediately spread the cells aver the surface ofthe plate) ‘4. “Clam shell” (lightly open) the lds and carefully pour 4-6 glass beads onto each plate '. Usea back-and-forth shaking motion (not swirling round and round) to move the glass beads across the entre surface ofthe pate(s). This should evenly spread the cll suspension allover the agar surface ‘&. When you finish spreading let the pte res for several minutes a allow the cell suspensions to become absorbed ino the agar. 44, To remove the glass beads, hold each plate vertically over a container clam shell the lower part ofthe plate and tapout the glass beads into the container. 15, Use another sterile transfer pipet to ad 100 of ell suspension from the plasm tube to each appropriste plate, 16, Immediately spread the cll uspension() as described in step 14 17, Wap the plates together wth ape and place the pats upside down, ther in the incubator oF at room temperature. Incubate them for approximately 24-36 hours in a 37°C incubator or 48-72 hours a room temperature a ata ga 9 ony $22 Bacterial Transformations