BME5, Introduction to Biotechnology Final Study Guide Lecture 4 Chromatographic methods used in enriching and purifying proteins Size

Exclusion Chromatogra hy ! se arates roteins "ased on size #small roteins enter ores$ Ion Exchange Chromatogra hy ! se arates roteins "ased on charge # roteins %ith highest o osite charge come o&& later$ '&&inity Chromatogra hy ! se arates roteins "ased on a"ility to "ind to s eci&ic molecules #anti"ody$ Preparing the crude extract; separating extract components (centrifugation, filtration, dialysis). (re aring the crude extract re)uires homogenization o& cells*tissues in a "u&&er Centri&ugation ! s ins do%n the homogenate into a li)uid and ellet+ most roteins are solu"le and in the li)uid Filtration ! remo,al o& cell de"ris, roteins, ions -ialysis ! tu"ing %ith ,ery small ore sizes to change solution rotein stored in SDS-PA ! S-S.(olyacrylamide Gel Electro horesis/ sodium dodecyl sul&ate denatures the roteins and co,ers them %ith negati,e charge to se arate "y size "estern #lotting (er&orms S-S.('GE and identi&ies rotein o& interest using anti"odies+ re ares an autorad $D- el !lectrophoresis Se arates roteins "ased on size #S-S.('GE$ and net charge Analy%ing the protein; &P'C, !dman Degradation, (ass Spectrometry 0igh (er&ormance Li)uid Chromatogra hy/ im ro,ed resolution and can se arate similar roteins Mass S ectrometry/ measures masses o& molecules and their arts Edman -egradation/ automated rotein se)uencing Lecture 5 Classes of micro)es 1hree domains o& li&e/ "acteria, archaea, eu2aryote #includes "acteria, archae, &ungi, rotozoa, ,iruses$ #acteria cell structure 3.5 um "acteria size+ contains chromosome and some lasmids, lasma mem"rane, cell %all made o& e tidoglycan, and ca sule sha es are cocci #s herical$, "acilli #rod$, s iral #cor2scre%$ Culturing )acteria; strea* and spread plates 4a id re roduction once e,ery 56 minutes under ideal conditions 1o culture, s read "acteria on agar late and allo% to gro% o,ernight at 78 Celcius to allo% roduction o& colonies Strea2 lates #strea2s to thin cultures$ and s read lates # ut a &e% dro s then s read$ +ransformation and electroporation 1rans&ormation/ '-----SS Electro oration/ uses electric shoc2 that results in transient ores %here -9' can enter cells #more e&&icient can trans&orm animal, lant or yeast cells, carry larger lasmids$ !xpression cloning; *no, mar*ers-components noted in lecture outline '----------'uciferase, ,hat it is, ,hat genes encode it and ho, it ,or*s as a reporter Luci&erase/ enzyme encoded "y Lux genes %hose roducts come together+ releases light in con,ersion o& Luci&erin to :xyluci&erin #cell glo%$ 4e orters signi&y that a gene ex ression has occurred Taq polymerase and cellulose 1a) olymerase/ -9' olymerase used in (C4, %ithstands high tem eratures used in cycling Cellulose/ '-----Anti)iotics; )asics, *no, one mechanism from the figure

'nti"iotics/ su"stances roduced "y one microorganism that inhi"its the gro%th o& "acteria+ don;t counter ,iral in&ections, made "y "acteria to inhi"it other "acteria L::< '1 1E=1B::< FIG>4E Smallpox .accine; !d,ard /enner0s ,or*, effecti.eness of the .accine 38?@ &irst ,accine roduced "y inAecting atients %ith li,e co% ox ,irus+ co% ox ,irus loo2s a lot li2e small ox and caused immune system to de,elo rotection against it Antigens, "hite #lood Cells and Anti)odies; *no, their roles in the immune response 'ntigens/ roteins or large car"ohydrates resent on the sur&ace o& "acteria, ,irus, &ungi that elicit anti"ody roduction (hagocytes/ engul& the antigen #macro hage$ Lym hocytes/ acti,ated "y antigen "inding+ 1 Cells Bcytotoxic "ind and destroy in,aders+ hel er secrete chemicals #cyto2ines$ that stimulate cytotoxic 1 and B CellsC and B Cells Bma2e anti"odies once acti,ated "y 1 0el er CellC 'nti"odies/ "ind to antigen and target in,ader Primary and secondary response; induction of acti.e immunity ,ith .accines (rimary 4es onse/ occurs the &irst time an indi,idual is ex osed to a certain antigen+ anti"ody roduction ta2es a &e% days and B Cells acti,ated to &orm (lasma and Memory Cells Secondary 4es onse/ occurs the su"se)uent ex osure to antigen and res onse is enhanced and utilizes Memory cells Dacti,e immunityE Classes of .accines 'ttenuated Faccines/ use li,e ,iruses*"acteria that ha,e "een modi&ied so they cannot re licate in host cell Inacti,ated Faccines/ uses inacti,ated or dead micro"es Su"unit Faccines/ uses only arts o& the ,irus or "acteria that elicit an immune res onse 1etro.irus life cycle 0a,e an 49' genome, ma2es a -9' co y o& 49' using re,erse transcri tase, and inserts -9' into host cell 2nfluen%a .iruses; &A protein0s role in infection and ho, changes in it can ma*e resistant strains. '-- M:444E Pulse3et and P4 ! -9' &inger rinting ro,ides a s eci&ic attern &or each micro"e (ulse9et/ net%or2 o& la"s using -9' D&inger rintingE to ra idly identi&y strains o& micro"es im ortant to u"lic health+ coordinated "y Centers &or -isease Control and (rotection (FGE G (ulsed.Field Gel Electro horesis Fersion o& agarose gel electro horesis modi&ied &or use %ith large ieces o& -9'+ electric &ield changed to &orce -9' &ragments to alter migration direction so smaller &ragments are se arated &rom larger ones Lecture @ Protoplast fusion and plant transgenesis (methods, tools and terminology) (roto last/ a lant cell treated to remo,e the cell %all #enzyme treatment$+ (roto last &usion roduces hy"rids "y com"ining t%o or more roto lasts (lant 1ransgenesis/ roducing genetically modi&ied lants through addition o& a transgene+ adding the Ad.antages of engineering chloroplast genes Antisense technology and the 4la.r Sa.r tomato 13Ai technology (*no, the mechanism) #e a)le to descri)e, and pro.ide an example, for one application of engineered plants #e a)le to descri)e one contro.ersy concerning use-culti.ation of engineered plants (ethods for engineering animals (*no, the details) 5noc*-out mice (ho, they are made-*no, the details, and ,hat they are used for) Producing monoclonal anti)odies in hy)ridoma cells (*no, the details)

&A(A response, ,hat it is and ho, it is addressed in anti)ody design SC3+ use in reproducti.e cloning, therapeutic cloning Lecture 8 6xidation-1eduction reactions, ,hat they are and ho, they are used in )ioremediation. . 4eductionG gains an electron :xidationG loses an electron, used in aero"ic and anaero"ic "iodegradation (icro)e classifications ,ith regard to oxygen effects on gro,th 4ertili%er-enhanced )ioremediation, )io.enting, )ioaugmentation and phytoremediation Ex situ )ioremediation +he Anammox process7 ,hat it is and ,hat it is used for 5no, the 8 main greenhouse gases and ,hich one is the largest contri)utor #e a)le to descri)e one adaptation strategy regarding preparing for climate change. Lecture H Polyploidy, ,hat it is, ho, it is created, ,hat it pro.ides to the organism (oly loidy G increased num"er o& sets o& chromosomes+ most animals* lants are di loid %ith gametes ha loid Created "y disru ting cell di,ision #cyto2inesis$ during meiosis or dis ermy #&ertilization o& an egg "y t%o s erms$ :rganisms gro% &aster and larger "ut usually sterile Lecture ? Phases of Clinical +rials (hase I/ 56.H6 eo le, is the treatment sa&e (hase II/ 366.766 eo le+ is the treatment e&&ecti,e and sa&e (hase III/ 3666.7666 eo le+ is the treatment e&&ecti,e on a range o& eo le and side e&&ects (hase IF/ has treatment "een %or2ing %ell and sa&e on the mar2et P D, Amniocentesis and C9S (G- G reim lantation genetic diagnosis 'mniocentesis/ er&ormed a"out 3@ %ee2s into regnancy+ collects amniotic &luid to test chromosome a"normalities '--S CFS G chorionic ,illus sam ling (er&ormed H.36 %ee2s into regnancy+ collects &etal tissue &rom de,elo ing lacenta %ith ris2 o& chromosomal mosaic 'ocate a chromosomal address such as :;<$.=. 2dentify the )and, region, arm and chromosome. Chromosome 5, ) arm, "and 35I7 #e a)le to recogni%e polyploidy and aneuploidy on *aryotypes and *no, the terms-conditions (oly loidy/ ha,ing an extra set#s$ o& chromosomes #ha loid, di loid, tri loid, tetra loid$ 'neu loidy/ addition or deletion o& a single chromosome &rom a di loid set #trisomy, monosomy$ CML and the (hiladel hia chromosome, ho% identi&iedI 4FL( analysis, %hat it is and ho% it is used in diagnosing Sic2le Cell 'nemia (at Bro%n;s Lym hochi and the 'm liChi CJ(456 test Entering the cell cycle, stages in,ol,edI Metastasic cancer ,s "enign tumor 4ece tor 1yrosine <inases and Cell Signaling #2no% the details$ 0E4 roteins/ %hat they are and the di&&erent ty es Khat cancer 0E45 is associated %ith, ho% it is targetedI 0emoglo"in structure and &unction 0emo ure and other hemoglo"in."ased thera eutics Gene thera y rocess

Gene thera y history Be a"le to descri"e one medical ris2*concern associated %ith gene thera y 1y es o& ,iral ,ectors #"e a"le to match each %ith a descri tion$ Gene thera y o& immune system disorders through altering hemato oietic stem cells Lecture 36 Stem cell ro erties 1y es o& stem cells*hierarchy 'dult , Em"ryonic Basics o& early em"ryo de,elo ment Characterizing adult and em"ryonic stem cells -irected di&&erentiation/ %hat it is and current status 1hera eutic cloning, '91 and i(S cells