Chng hai

C s ha sinh v di truyn hc ca cng ngh sinh hc vi sinh vt

I. Phn loi cc sn phm Cc cht c sn xut bng con ng ln men nh vi sinh vt rt a dng. tin cho nghin cu v ng dng th phi tin hnh phn loi sn phm ln men cng nghip da vo tiu chun sinh l sinh ha trao i cht ca vi sinh vt. Chnh v vy cng tc phn loi sn phm l vic lm cn thit ca cng ngh vi sinh. 1. Sinh khi (vt cht t bo) Vic tng hp sinh khi t bo l qu trnh thc hin sn xut protein vi sinh vt. Qu trnh ny thc cht l qu trnh sinh trng ca vi sinh vt. Qu trnh ny pht trin nhng c ch iu ha trao i cht m bo mt phn ln n mc cho php cc cht dinh dng cung cp s c s dng vo qu trnh tng hp cc thnh phn ca t bo. 2. Sn phm trao i cht + Sn phm ca qu trnh ln men: Ln men l mt trong nhng con ng ca qu trnh trao i nng lng. Ngoi vic cung cp nng lng cho t bo vi sinh vt, cn cung cp cc sn phm c gi tr cho con ngi nh: ethanol, acid acetic, acid lactic, acid propionic, kh methane, cc c cht giu hu c khc... + Cc cht trao i bc 1: L nhng vin gch cu trc nn vt cht ca t bo, trong s cc cao phn t sinh hc th y l nhng cht c phn t lng thp: cc amino acid, nucleozide, nucleotide, ng. Ngoi ra, cc cht trao i bc 1 cn l sn phm ca qu trnh trao i cht trung gian nh acid hu c ca chu trnh Tricarboxylic. + Cc cht trao i bc 2: L nhng cht trao i c phn t lng thp, khng gp trong c th vi sinh vt. Nhng cht ny khng c chc nng chung trong trao i cht ca t bo nh: cc cht khng sinh, cc c t, cc cht c hot cht kch thch sinh trng nh gibberellin. + Enzyme: L nhng protein xc tc c s bin i cc cht ca t bo. Mi t bo vi sinh vt c khong 1000 loi enzyme khc nhau vi s phn t ln n 106, gm enzyme ni v enzyme ngoi bo nh: amylase, proteaea, cellulase... trong enzyme ni bo chim a s. 3. Cc sn phm ca s chuyn ha cht
t bo vi sinh vt Sn phm Tin sn phm

T bo vi sinh vt thng qua h enzyme ca mnh ng vai tr xc tc cho cc phn ng chuyn ha cc cht. V mt l thuyt nhng phn ng ny c th xy ra nh nhng xc tc ha hc no . Tuy nhin cc qu trnh ny i khi khng thc hin c iu kin bnh thng, m ch thc hin iu kin c bit (nhit , p sut, m) thch hp cho qu trnh chuyn ha, trong trng hp ny ngi ta chuyn sang sn xut bng cng ngh vi sinh vt. V d t ethanol chuyn n acetic acid phi dng chng Acetobacter, Acetomonas chuyn ha.

II. Mi quan h gia sinh trng ca vi sinh vt v s to thnh sn phm Trong iu kin nui cy tnh, qu trnh sinh trng ca vi sinh vt tri qua 3 pha, c biu din bng th (hnh 1).
Sinh trng v to sn phm Giai on dinh dng Giai on c th

Thi gian

Thi gian

Hnh 1: Mi quan h gia sinh trng v to thnh sn phm ca vi sinh vt Trong iu kin nui cy ton b qu trnh sinh trng ca vi sinh vt gn lin vi s thay i theo thi gian. Trong mi trng, cc cht dinh dng theo thi gian s gim, v tng ng s lng t bo vi sinh vt s tng ln, ng thi hot tnh trao i cht ca t bo cng thay i. Lc ny cc sn phm trao i cht c th c vai tr khc nhau i vi t bo. C th tm chia sn phm ra thnh 2 loi sau: + Loi sn phm m s hnh thnh ca n gn lin vi sinh trng ca vi sinh vt, nh cc cht trao i bc 1: Cc enzyme, cc sn phm ca qu trnh ln men. S tng hp loi sn phm ny xy ra trong thi gian sinh trng v cn c th tip din sau khi sinh trng kt thc. + Loi sn phm m s hnh thnh ca chng khng cn thit cho sinh trng ca vi sinh vt, nh cc cht trao i bc 2. S tng hp cc cht ny xy ra sau khi sinh trng kt thc ( vo pha tnh. Gio trnh vi sinh vt i cng). S to thnh sn phm trong giai on ny c gi l sn xut hay giai on c th, hoc giai on dinh dng. Tuy vy cng c nhiu sn phm m s hnh thnh ca n khng nm trong hai giai on trn, tm gi l dng trung gian, v d s hnh thnh amino acid, mc d sn phm ny c hnh thnh giai on dinh dng, nhng n vn cn tip din sau khi sinh trng kt thc, v qu trnh tng hp cc amino acid tip din trn c s ca mt sai hng v iu ha trao i cht tng hp. T cho thy, trong cng ngh ln men i hi nh sn xut phi bit sn phm ca mnh cn thu c sinh ra giai on no ca qu trnh nui cy, ng thi phi bit tm mi bin php ti u ha qu trnh nui cy cho hiu sut to sn phm cao nht. Ngha l tm ra iu kin nui cy m bo cho vi sinh vt t trng thi sinh trng, pht trin ti u. Trong cng ngh ln men, ch cui cng cn phi t l: t c cht ban u vi mt dung tch ni ln men nh nht, trong thi gian ngn, c th thu hoch sn phm mong mun vi nng sut cao nht. Nh vy mi gim c gi thnh. Mt quy trnh cng ngh nh vy mi l hon thin.

III. Nhng nguyn tc iu ha trao i cht Trong hot ng sng ca mnh, vi sinh vt to cc sn phm trao i cht v cc thnh phn cu to nn t bo ch mc cn thit cho sinh trng, pht trin, sinh sn duy tr loi. C ngha l trong t nhin khng c s sinh sn d tha cc sn phm trao i cht bc 1, 2. 1. iu ha hot tnh enzyme nh s km hm do lin kt ngc Ngi ta nhn thy: sn phm cui cng ca qu trnh sinh tng hp mt cht c kh nng gy ra s c ch qu trnh tng hp ca chnh n. Sn phm cui cng d c vi sinh vt tng hp nn hay thu nhn t mi trng ngoi, khi nng d tha so vi nhu cu ca c th vi sinh vt s nh hng n enzyme u tin trong chui sinh tng hp. S chui cc phn ng sinh ha xy ra tng hp cht (X):
(a) (b) (c) A B C X

Enzyme u tin (a) l mt enzyme d lp th, n c c im cu trc hnh khng gian khi c mt sn phm cui cng nhm gim bt hot tnh xc tc ca mnh. enzyme ny, ngoi v tr gn vi c cht A (trung tm xc tc), n cn c mt hay nhiu v tr gn vi sn phm cui cng X gi l trung tm d lp th. Trung tm xc tc v trung tm d lp th tch bit nhau v khng gian v cu trc. Trng thi hot ng ca enzyme ny c c trng ch n c kh nng gn vi c cht A v nu bn cnh c cht A cn c s hin din ca X mc d tha so vi nhu cu ca c th vi sinh vt, th s xy ra s bao vy ca trung tm d lp th, lm cho trung tm xc tc b bin i cu hnh khng gian n mc khin cho enzyme (a) khng th gn c vi c cht A, m ch gn vi X. Nh vy enzyme (a) s khng c hiu lc trong vic chuyn ha A thnh B. Chui sinh tng hp X s b gin on. Khi X s b gim s lng. S iu ha ny mc enzyme. 2. S cm ng v c ch qu trnh tng hp enzyme Trong khi nui cy vi sinh vt c mt cht kh ng ha, vi sinh vt phi tit vo mi trng mt hoc vi enzyme tng ng phn hu c cht thnh c cht c th ng ha c. Enzyme c hnh thnh ny c gi l enzyme cm ng. C cht kch thch qu trnh ny c gi l cht cm ng. S cm ng v c ch qu trnh tng hp enzyme vi sinh vt c Hc thuyt operon ca F. Jacob v J. Monod tm ra nm 1966. Hc thuyt operon gip lm sng t c ch iu ha chng trnh lm vic ca b gen i vi qu trnh tng hp protein - enzyme. + Nhm gen cu trc: Nhm gen ny m bo vic m ha cu trc ca cc phn t protein - enzyme. Cc gen ny thng xp lin nhau. Trong trng hp Lac. coli, cc gen cu trc gm ba gen k hiu l A, B v C. - Gen A m ha th t amino acid ca -galactosidase. - Gen B m ha cu trc ca enzyme thm thu galactosidepermease cn thit cho qu trnh vn chuyn lactose vo t bo. - Gen C m ha cu trc ca enzyme thiogalactoseacetyltransferase. C ba gen cu trc ni trn to thnh mt n v phin m.

+ Gen iu khin (Operator): L on DNA nm k bn nhm gen cu trc, k hiu l O. Nh tc dng gn vi cht c ch, operator lm vic nh mt cng tc ph trch vic ng m hot ng ca nhm gen cu trc. + Gen khi ng (Promoter): L DNA nm k pha trc operator l ni gn enzyme RNA polymerase, enzyme ny xc tc cho qu trnh tng hp RNA thng tin ca nhm gen cu trc. Khi cht c ch gn vo operator th phn t RNA polymerase b cn tr, khng di chuyn dc theo mch khun DNA, dn n cc gen cu trc b km ch v khng to c protein cng nh enzyme tng ng. Do v tr v chc nng nh vy nn c gi l gen Promoter (gen khi ng). + Gen iu ha (Regulator): Gen ny chu trch nhim m ha vic tng hp nn mt protein c bit ng vai tr cht c ch. Thng n ch c tng hp vi mt lng khng ng k trong t bo (khong 10 - 20 phn t/t bo). c im ca cht c ch l mt protein bin cu oligomer c hai tm c th. Hai tm ny lm cho cht c ch c kh nng hoc gn vi cht cm ng hoc gn vi operator. Nu cht c ch c i lc ln vi operator, th thng gn vo operator. * C th khi qut ha iu kin cm ng nh sau: - Trn nhim sc th ca t bo phi c nhng gen tng ng vi cc enzyme s c hnh thnh. - Cc nguyn liu xy dng phn t enzyme: cc amino acid v cc hp phn ca nhm ngoi. - Nng lng cn thit cho vic hnh thnh cc lin kt. - Cht cm ng. Theo F. Jocob v Monod, th d c ba iu kin trn m khng c cht cm ng th enzyme cm ng cng khng c to thnh. Nh vy hnh thnh mt enzyme cm ng phi hi t bn iu kin: gen, nguyn liu xy dng, nng lng v cht cm ng. 3. iu ha tng hp enzyme nh s kim ch bng sn phm cui cng v s phn gii kim ch Nu chng ta k hiu X l sn phm cui cng ca mt chui sinh tng hp, th thy rng X c tc dng c hiu vi cht c ch (cht do gen iu ha tng hp nn). Khi mi trng c hin tng d tha X so vi nhu cu ca t bo, X s gn vi cht c ch, lm thay i cu hnh khng gian ca cht c ch, lm cho cht c ch c kh nng gn vi operator (cn gi l hot ha cht c ch). Do vy gi cht X l cht ng km hm. Khi cht c ch gn vi operator s lm ngng tr qu trnh phin m, c ch operon, dn n enzyme khng tng hp c, khi vic sn xut X b gin on. Trong khi t bo vn tip tc s dng cht X, khin cho cht ny b gim ti mc khng p ng nhu cu ca t bo. Lc y s xy ra qu trnh gii phng s kim ch operon ni trn, v do thiu cht X, cht c ch lc ny s thiu mt yu t hot ng ha hc, do khng c kh nng gn vi operator, iu ny dn n gii phng operon, dn ti cc enzyme c tng hp v vic sn xut X s c tin hnh tr li. l hin tng gii kim ch. 4. iu ha tng hp enzyme nh s kim ch d ha Trong nui cy vi sinh vt c nhiu ngun c cht, trc ht xy ra vic tng hp cc enzyme xc tc cho s phn gii c cht d s dng nht. S tng hp cc enzyme xc tc phn hu cc c cht khc b c ch bi s kim ch d ha. V d: Trong mi trng nui cy c hai ngun carbohydrate l: glucose v lactose. Trc tin vi sinh vt s hnh thnh cc enzyme phn gii

glucose. S cm ng tng hp enzyme phn gii lactose - galactosidase b c ch bi s kim ch d ha. C ch kim ch d ha c nghin cu kh chi tit vi khun E. coli vi vic iu ha tng hp enzyme - galactosidase. Nu trong trng hp carbohydrate, glucose l ngun c cht c s dng thch hp nht, v vy khi c mt glucose th nhiu enzyme khc ca qu trnh d ha cng nh trao i cht trung gian khng c tng hp. Ngi ta gi hin tng ny l hiu ng glucose. + Khi mi trng khng c glucose, c lactose: Mi trng khng c glucose, s dn n tch lu mt lng ln AMPv (adenosine monophosphate vng). Lc AMPv s phn ng vi mt protein nhn k hiu l CAP - (catabolite activato rprotein). Ngi ta cn gi hin tng ny l vng Promoter b cht y. Chnh s cht y ny l tin cho s hot ng ca enzyme RNA polymerase, c ngha l s thc y s c hot ha nh s cht y. ng thi trong mi trng cn c mt lactose, lactose s ng vai tr l mt c cht cm ng, n s phn ng vi cht c ch, lm thay i cu hnh khng gian ca cht c ch to phc h c ch - cht cm ng. iu ny khin cho cht c ch khng gn c vi operator. Nh vy Lac-operon s c gii phng. + Khi mi trng c glucose, c lactose: Trong mi trng nu ngoi lactose cn c b sung glucose, th lc y hm lng AMPv s b gim i, hu qu l CAP khng to c phc h vi AMPv, do khng c s cht y Promoter. Lin i RNA polymerase s khng c m u hot ng hoc nu c m cng rt yu. V vy ngay c khi c mt c cht cm ng (lactose) cng khng c s tng hp RNA thng tin- c ngha khng c s to thnh enzyme. + Khi mi trng khng c glucose v khng c lactose: Nu trong mi trng khng c c hai glucose v lactose, th cht c ch s gn vo operator nn operon vn b phong ta. Do RNA thng tin khng c tng hp. Khng c s tng hp protein- enzyme. IV. Nhng sai hng di truyn ca iu ha trao i cht v hin tng siu tng hp Nhng c ch iu ha ni trn gip cho c th vi sinh vt m bo c hot ng sng ca mnh tin hnh mt cch nhp nhng trn c s tit kim nguyn liu, nng lng mt cch hp l. Tuy nhin, nu mi vi sinh vt u c hot ng sng bnh thng th khng c l do g quan tm c bit n chng. Trong hot ng sng ca vi sinh vt chng lun tit ra cc sn phm no , m nhng sn phm ny li rt cn thit cho con ngi. So vi nhu cu cho hot ng sng ca vi sinh vt, nhng sn phm chng tng hp c chc chn l d tha lng ln. Ngi ta ni: Nhng c th vi sinh vt ny c kh nng siu tng hp mt cht no . Vi s pht trin ca khoa hc, hin nay con ngi to c rt nhiu chng ging vi sinh vt c kh nng siu tng hp cc cht. y l kt qu ca qu trnh chn lc nhn to vi cc phng php gy t bin. Nhng chng t bin ny c nhng sai hng di truyn rt ng c quan tm.

1. Cc chng t bin mt i c ch iu ha hot tnh enzyme bng sn phm cui cng Li dng c ch iu ha hot tnh enzyme bng sn phm cui cng, ngi ta dng t bin lm hng trung tm d lp th ca enzyme (a), lm cho n mt kh nng gn vi cht X nhng bn thn enzyme (a) vn cn hot tnh xc tc i vi c cht A (xem s chui cc phn ng sinh ho trang 14). Do vy khi c mt cht X sn phm cui cng vi s lng d tha so vi nhu cu ca vi sinh vt, enzyme (a) vn xc tc chuyn A thnh B, dn n cht X - vn c tip tc tng hp. 2. Cc chng t bin c s sai hng c ch iu ha tng hp enzyme Ngi ta dng cc cht gy t bin lm sai hng c ch iu ha tng hp enzyme. C th l ng chm n gen iu ha (Regulator) - gen chi phi to nn cht c ch, dn n s sai hng ca cht c ch hoc thm ch c th ph hu qu trnh tng hp cht c ch. Hay c khi t bin ng chm n gen iu khin (Operator) lm cho gen ny mt kh nng gn vi cht c ch. Kt qu ca tc ng trn l ngay c khi mt cht no c nng d tha so vi nhu cu ca vi sinh vt, cc enzyme cn thit cho s tng hp ca chng vn c hnh thnh v cc cht ny vn c tip tc tng hp trong t bo. V. ngha ca k thut di truyn tm c chng vi sinh vt theo s mong mun, con ngi tm cch tc ng vo cc quy lut iu khin qu trnh trao i cht ca vi sinh vt. Vic to nn nhng chng t bin ny da trn c s ca nhng hiu bit v quy lut di truyn v bin d ca vi sinh vt, da trn nhng kinh nhim ca cng tc lai to ging. Nhng thnh cng ca cng ngh vi sinh cho php chng ta ch ng to c cc DNA ti t hp trong iu kin in vitro. Cng hiu thm v sinh hc phn t, di truyn hc v cng ngh gen. C th ni rng ngy nay con ngi c th chuyn nhng on gen t sinh vt ny sang sinh vt khc c s khc bit rt ln v di truyn, hay ni cch khc l c th ct b hng ro gia cc loi do to ha gy dng nn cn tr s giao phi khc loi, nhm bo tn tnh c trng ca loi. Tuy nhin y mi ch l bc u v cng ngh gen. VI. Nhng hiu bit v chuyn ti gen y l vn mi v rt phc tp, chng ta tm hiu khi qut hai vn sau: + Nhng cu trc tham gia chuyn ti gen. + Qu trnh thun ha v chuyn ti gen nh vi sinh vt. 1. Nhng cu trc tham gia chuyn ti gen gi l th mang (vector) Cc vector chuyn ha gen phi tho mn nhng yu cu ti thiu sau: - L cc on phn t DNA c kh nng t sao chp tch cc trong t bo ch, tn ti c lp trong t bo khng ph thuc s sao chp ca b gen t bo ch. - C kch thc nh. Cng nh cng tt, v vector c kch thc cng nh th cng d xm nhp vo t bo vi khun khc v cng c sao chp nhanh, c hiu qu. - C trnh t nhn bit duy nht ca cc enzyme gii hn (RE). - C kh nng cha mu DNA ngoi lai.

- C gen nh du, tc l c kh nng biu hin ra bn ngoi d nhn bit. Gen nh du c gi l Marker, ngi nghin cu nhn bit v d dng tch t bo c cha gen cn chuyn ti ra khi qun th vi khun. V d: Ngi ta thng dng gen chi phi tnh trng khng vi cht khng sinh lm gen nh du. Trong mi trng c cha khng sinh, th ch nhng vi khun c mang gen khng khng sinh mi sng st. Trong s cc cu trc c s dng tham gia chuyn ti gen c th k n plasmid, phage, cosmid, YAC..., m ph bin nht l plasmid v phage. + Plasmid: t bo Prokaryote, c th l vi khun, qua knh hin vi in t c th quan st c cht nhn l phn t DNA nguyn vn c dng vng trn hnh chic nhn. l nhim sc th - ni cha nguyn liu di truyn ca t bo. Cng vi nhim sc th cn c cu trc hnh nhn nh hn ngi ta gi l plasmid. em tch plasmid di dng tinh khit tm hiu cu trc v tnh cht ca n, th thy plasmid c cu to t DNA c kh nng t nhn i mt cch c lp v tn ti mt cch c lp vi b gen ca vi khun. V vy ngi ta coi plasmid l phn t di truyn nm ngoi b my di truyn ca vi khun. nhm Eukaryote, ngi ta mi pht hin ra c plasmid nm men. Ngi ta thy plasmid c hng lot c im ring l: - Plasmid tham gia vo c ch ti t hp gen ni bo. - Plasmid c kh nng di chuyn t mt vi khun ny sang vi khun khc. - Plasmid c kh nng vn chuyn gen. - Plasmid c kh nng sinh sn cc nhanh v c hot tnh mnh. + Bacteriophage (Phage hay thc khun th): - Phage c kch thc cc k nh qua c mng lc vi khun. - Phage th hin tnh c i vi vi khun qua chu trnh sinh sn gy c ca phage, c kh nng xm nhp vo t bo vi khun, nh ch qu trnh trao i cht ca vi khun v ly nguyn liu t vi khun xy dng nn cc thnh phn ca n k c nguyn liu di truyn. Do vy hnh thnh nhng on DNA ti t hp, bao gm cc on gen ca phage v ca vi khun. Qua thc nghim cho thy, vic s dng phage lm th mang c nhiu u im hn so vi plasmid, v phage c nhng c im gip s xm nhp vo t bo vi khun hiu qu hn nhiu so vi s chuyn plasmid vo vi khun. Hn na phage, kch thc ca on DNA n c th tip nhn ln hn nhiu so vi s tip nhn ca plasmid. 2. Qu trnh thun ha v chuyn ti gen nh vi sinh vt Thun ha gen l qu trnh bt gen phi lm vic theo mun ca con ngi. Qu trnh ny rt phc tp, i hi c nhng hiu bit su sc v c tnh ca gen v k thut phn t. Trong k thut phn t, th vai tr ca enzyme l quan trng nht. Enzyme y gm nhiu loi: nuclease, ligase, polymerase (DNA polymerase v RNA polymerase). 2.1. Cc enzyme phn ct DNA (RNA) c gi l nuclease. Cc nuclease gm hai nhm: endonuclease v exonuclease. - Endonuclease l nhng enzyme ct DNA gia phn t, cn exonuclease ct t hai u mt ca phn t DNA. Trong nhm endonuclease c cc enzyme gii hn (RE - restriction

enzyme), nhng enzyme ny c cc nh khoa hc c bit quan tm v n c c tnh rt qu, c tnh c hiu rt cao, n ch ct DNA mch kp nhng ch nht nh ch khng linh ng. Nhng im nhn bit ca enzyme ny thng c trnh t 4 - 6 cp nucleotide i xng o ngc nhau c gi l palindrome. Mi RE c trnh t nhn bit c trng. - Hin nay ngi ta pht hin c 500 loi RE, trong s c hn 100 loi RE c bn trn th trng. Mi ch pht hin RE cc nhm sinh vt nhn s Prokaryote, cn nhm nhn tht cha pht hin thy. Do c tnh c bn ca cc RE l c kh nng nhn bit v ct mt trnh t xc nh trn phn t DNA, m ngi ta chia RE ra thnh cc loi sau: Loi I: Khi enzyme nhn bit c trnh t, n s di chuyn trn phn t DNA n cch khong 1000 - 5000 nucleotide v ct. Loi II: Enzyme nhn bit c trnh t v ct ngay ti v tr . Loi III: Enzyme nhn bit mt trnh t v ct DNA v tr cch khong 20 nucleotide. Trong s 3 loi RE trn, th loi II c quan tm v s dng nhiu trong lnh vc phn t. 2.2. Cc enzyme gn Trong nhm ny ngi ta s dng ph bin cc enzyme xc tc s hnh thnh lin kt ni 2 on DNA (DNA ligase) hay RNA (RNA ligase). + Trong cc ligase thng s dng phi k n: - E. coli DNA ligase: Enzyme c trch ly t trc khun E. coli v xc tc phn ng ni hai trnh t DNA c u so le. - T4 DNA ligase: C ngun gc t phage T4 xm nhim E. coli, enzyme ny c cng chc nng vi ligase trch t E. coli ni trn, nhng c bit cn c kh nng ni hai trnh t DNA u bng, nn ligase c chung nht trong k thut to chng. - T4 RNA ligase: Enzyme c trch ly t phage T4 xm nhim E. coli, xc tc qu trnh ni hai trnh t RNA bng lin kt phosphodiester. + Qu trnh thun ha v chuyn gen c th chia thnh 3 bc sau: - Thu nhn gen cn chuyn: C th thu nhn gen trc tip t b gen bng cch lc c hc hay ct bng RE, hoc tng hp ha hc theo trnh t nucleotide bit ca gen hoc sinh tng hp gen t RNAm ca n nh enzyme phin m ngc reverse. - To vector ti t hp: Sau khi c dng thun khit ngi ta gn n vo cc vector ti t hp. Trc tin ngi ta ct vector v gen bng cng mt loi RE ti nhng trnh t nhn bit ca n. Nh vy hai u on gen cn chuyn v hai u vector b ct c nhng u dnh (trnh t DNA mch n b sung nhau), trn ln DNA cn chuyn v vector cc u dnh s bt cp vi nhau. Dng ligase hn dnh li, ngi ta c vector ti t hp. - Chuyn vector vo t bo nhn, lm clone ha cc gen (tc l nhn bn gen) va to nn trong t bo nhn, chn ra dng t bo cha gen mong mun. Bc tip theo ca qu trnh chuyn ti gen l chuyn cc vector ti t hp to thnh trong iu kin in vitro vo t bo nhn thch hp. i vi vector l plasmid, y l qu trnh bin np, c h tr bng nhiu cch khc nhau. i vi vector l phage, n c kh nng t ng thc hin ti np vi hiu sut cao hn nhiu. Tu i tng nhn gen v yu cu c th m ngi ta la chn p dng phng php no hiu qu nht: vi khun E. coli, x l t bo bng CaCl2 nhit thp lm cho mng t bo tr nn d tip nhn plasmid. mt s vi khun khc v nm men phi x l to dng t bo trn (protoplast) bin np mi thc hin c.