BI 1: GING VI SINH VT V CI TO GING VI SINH VT

CHNG TRNH HC

MN K THUT DI TRUYN VI SINH VT

BI 1: GING VI SINH VT V CI TO GING VI SINH VT1. GING VI SINH VT

1.1. Sinh l vi sinh vt: Gm 4 pha:

Pha lag (pha tin pht): Giai on ny c tnh t lc bt u nui cy n lc bt u thy s sinh trng v pht trin nhanh u giai on ny, vi sinh vt cha tin hnh sinh sn m ch xy ra qu trnh thch nghi vi mi trng nui cy. Thi gian pha ny ph thuc vo:

Tui sinh l ca ging vi sinh vt

Cht lng thnh phn mi trng.

Pha log (Pha cp s): c biu hin r nt bi tc sinh sn ca vi sinh vt t cc i. T bo va sinh sn mnh, va tng sinh khi theo cp s 2eq \l(\o\ac(n, )). Trong pha ny vi sinh vt tin hnh tng hp enzyme vi s lng v cht lng cao.

Pha cn bng (Pha n nh): Cc cht dinh dng trong mi trng nui cy gim i r rt. Cc cht to ra do qu trnh trao i cht c tch lu trong mi trng rt ln. Sinh khi chung trong pha ny t c l cao nht trong qu trnh nui cy.

Pha suy vong: S lng t bo cht tng nhanh v:

Cht dinh dng trong mi trng gim dn n ht.

T bo n giai on gi.

Sn phm trao i cht trong mi trng qu nhiu, gy c ch qu trnh trao i cht ca t bo.

1.2. Vai tr ca ging vi sinh vt

Trong cng ngh vi sinh vt, ging ng vai tr quyt nh.

1) Ging quyt nh nng sut sinh hc ca nh my.2) Ging quyt nh cht lng ca sn phm sinh hc.3) Ging quyt nh vn u t cho sn xut.4) Ging quyt nh gi thnh sn phm.

1.3. Cc yu cu v ging vi sinh vt

1) Ging vi sinh vt phi cho ra sn phm m ta mong mun. Sn phm ny phi c s lng v cht lng cao hn cc sn phm ph khc.2) Ging phi cho nng sut sinh hc cao.3) Ging vi sinh vt phi c kh nng ng ho cc nguyn liu r tin v d kim ti a phng ni nh my ang hot ng.4) Sn phm ca qu trnh ln men do ging ang p dng trong sn xut phi d thu nhn v tinh sch.5) L nhng vi sinh vt thun khit, khng c nhim cc vi sinh vt l, phi n nh v fenotype v genotype.6) Tnh thch nghi cao trong iu kin ln men cng nghip (n nh nhit , pH, p sut thm thu, hot tnh ca nc)7) Ging phi c tc sinh sn v pht trin mnh trong iu kin mi trng cng nghip ( Tnh cnh tranh v c ch s pht trin ca vi sinh vt l.8) Tc trao i cht mnh nhanh to ra sn phm mong mun.9) Ging phi n nh trong bo qun v d dng bo qun.

1.4. Phn lp ging

1.4.1. Phn lp ging trong t nhin

Thin nhin l ngun v tn cho phn lp cc loi ging vi sinh vt, tuy nhin vi sinh vt phn b trong t nhin khng u. Chng thng tp trung nhng ni giu cht dinh dng v iu kin pht trin thun li.

Nguyn tc c bn: u tn ti mt c cht no th c nhiu vi sinh vt phn hu c cht .

c im ca vi sinh vt trong t nhin:

Tp trung nhng ni giu cht dinh dng v iu kin pht trin thun li.

t khi cho nng sut sinh hc cao nn mun c ging tt phi ci to ging ny.

Kh p dng quy m cng nghip.

Vic chn ging t iu kin t nhin thc s l mt cng vic tm kim cc chng t bin t nhin c tnh trng di truyn tri ph hp vi mc tiu ra. Tuy nhin vic chn ging ny gp nhng kh khn sau:

Nhng bin d thng ngu nhin khng nh hng, chng li lun ln vi nhng vi sinh vt khng bin d.

Cc th bin d ny c th l thng bin v c th l t bin di truyn.

S lng t bin di truyn trong thin nhin khng nhiu.

Vic tuyn chn trong iu kin t nhin ch yu l to ging ban u sau phi nng cao cht lng ging.

Tin hnh phn lp:

Xc nh v tr phn lp ging (Tho iu kin: tp trung kh cao v mt vi sinh vt m ta quan tm)

Cn 1gram hay 1ml em phn lp.

Chun b ng nghim cha mi trng tp trung: Loi dn nhng vi sinh vt m ta khng quan tm.

Pha long ( cy vo mi trng tp trung.

Mi trng c hiu: ch cho vi sinh vt cn phn lp pht trin.

nh danh ging cn phn lp (dng SHPT, Kit, gi mu nh danh)

1.4.2. Phn lp ging trong nh my

Vi sinh vt trong iu kin sn xut c nhng u im:

D thch nghi vi iu kin sn xut.

Cho nng sut v cht lng sn phm sinh hc cao.

Trong nc thi v cht thi nh my c mt vi sinh vt cn quan tm nht.

Tin hnh phn lp:

Xc nh a im v mu vt em phn lp iu kin sn xut bao gm: (dch ang tin hnh ln men, nc thi ca nh my, cht thi ca nh my.)

Cc bc tip theo ging phn lp ging trong t nhin.

1.4.3. Hot ho ging

Mi trng bi dng.

Sng lc ging.

Rt gn pha lag.

Chiu tia t ngoi, lazer (qut nhanh tng qu trnh trao i cht)

Khi ly ging cc Ngn hng ging cn quan tm n l lch ging v yu cu pht trin ging.

1.5. To ging vi sinh vt

Nguyn tc: u c c cht th c vi sinh vt phn gii c cht .

Mu vi sinh vt:

T nhin

Nh my

Ngn hng ging vi sinh vt (Trung tm gi ging vi sinh vt chun HQG HN)

1.5.1. V d 1: Phn lp ging bifidobacteria t phn su tr em.

Trn mi trng RCA, khun lc bifidobacteria c dng hnh trn, mu trng sa, bng lng, c mp v hi li, ng knh khong 1 - 1,2 mm.

c im hnh thi t bo, kh nng di ng, cch sp xp t bo v trng thi Gram ca chng c phn lp bifidobacteria sp xp t bo dng ch Y.

Kh nng nh hng ca oxy ln chng vi sinh vt c phn lp.

Kho st c im sinh ho.

Th nghim catalase: (-), khng thy hin tng si bt.

Th nghim decarboxylase.

Th nghim kh nng ln men cc ngun carbon.

Xc nh chng vi sinh vt phn lp bng phng php PCR.

Primer: g -Bifid - F

g - Bifid - R(549 - 563 bp)

1.5.2. V d 2: Phn lp ging Lactococcus lactis ng dng sn xut ch phm Bacteriocin.

S chn Lactococcus lactis da vo c im hnh thi: khun lc trn, mu trng sa

Chn mt s c im sinh ho in hnh sng lc:

Kh nng sinh acid lm tan CaCOeq \l(\o\ac( ,3)) trn mi trng Carbonat Agar.

Kh nng sinh acid lactic.

Kh nng tng trng trn mi trng thch su.

Kh nng ln men ng.

Kh nng sinh tng hp Bacteriocin ( Bacteriocin c chc nng dit khun to thnh vng trn trong khng khun (vng cng ln th hot tnh Bacteriocin ln)

nh danh bng b lit: API50CHL.

1.5.3 V d 3: Phn lp ging Bacillus subtilis ng dng trong sn xut cc ch phm probiotic.

c im hnh thi.

c im vi th.

c im sinh ho.

Quy trnh nh danh.

Ly trch DNA.

PCR khuch i rDNA 16s.

Tinh sch

PCR gii trnh t ( i chiu kt qu vi ngn hng gen.

i chiu ti ngn hng gen (mc tng ng)

2. CI THIN GING VI SINH VT

2.1. Hun luyn thch nghi

Nguyn tc 1:

Tt c vi sinh vt u c kh nng thch nghi vi iu kin sng ca chng rt cao.

Hun luyn thch nghi vi iu kin sn xut cng nghip.

Tip cn vi nng sut cao nht ca n trong t nhin.

Bt n phi thay i qu trnh trao i cht.

Hun luyn thch nghi nng cao cht lng ging.

Nguyn tc 2:

Khi to c kh nng thch nghi ca ging vi sinh vt i vi mt tc ng mi trng no phi lun lun duy tr tc ng mc to ra thch nghi. Nu ph b tc ng ny hoc lm thay i tc ng ny s ph b tnh thch nghi. Khi ging rt d tr li trng thi ban u ca n. Thc cht ca tnh thch nghi l thng bin, thng bin th khng di truyn. Do c im ny khng bn. Cc bc tin hnh to ging thch nghi c thc hin nh sau:

Lp bn mc tng dn ( C cht kh...) hay gim dn (pH...) ca yu t tc ng.

Nui ging trong nhng iu kin tng dn hoc gim dn theo thang bc tng dn hay gim dn (Thc hin nhiu ln)

Gi ging trong iu kin to c tnh thch nghi .

2.2. Thay i c tnh di truyn

2.2.1. Phng php di truyn c in: bao gm lai ging v k thut to t bin nhn to.2.2.1.1. Phng php K thut lai ging: + To t bo trn

+ Ti to v

+ Nhn ln

u im: d thc hin

Nhc im: gii thiu s pha trn c tnh di truyn ch trong mt loi nht nh.

2.2.1.2. Gy t bin nhn to: l k thut to ra nhng t bin im (NST, gen...)

u im: ph v gii hn loi

Nhc im: khng nh hng.

HNO2 Thuc nhm cc tc nhn gy t bin ha hc, gy nn cc bin i v mt ha hc cc base nit ca DNA.

Tc ng ca HNOeq \l(\o\ac( ,2)) l gy t bin im.

Di tc dng ca acid ny, adenin tr thnh hypoxantin, guanin tr thnh xantin v cytozin tr thnh uracil ( cp A T c thay th bng cp G X.

Khi G b kh amin tr thnh xantin th vic thay th khng lm ri lon (xantin tng t guanin), c xu hng kt i vi cytozin. Xantin c th b hon ton loi b.

Tia t ngoi

Tc nhn t bin vt l c s dng rng ri nht trong di truyn hc.

Di tc dng ca tia t ngoi, VSV b mt hot tnh nhiu nht 260 nm.

bc sng 260 nm, vic mt hot tnh DNA cng t ti cc i.

Pha n nh, t bin xut hin t hn pha log (tng trng).

Hin tng quang phc hi: sau khi lm mt hot tnh bng tia t ngoi, em chng chiu nh sng bnh thng (320 480 nm) th t 50 80% t bo c phc hi hot tnh.

( sng lc th t bin trong bng ti.

2.2.2. Phng php di truyn hin i

L k thut ti t hp gen (Phng php chuyn gen v to dng)

Khng b gii hn loi v cc bin i di truyn c tnh nh hng.

K thut di truyn hin i da vo nhng pht hin:

Plasmid, enzyme ct gii hn , enzyme lin kt.

Hai xu hng thc hin ti t hp:

Chuyn gen vi sinh vt sang ng vt hay thc vt nhm ci tin cac tnh trng hoc lm tng thm tnh trng m ta cn cho c th ng vt hay c th thc vt.

Chuyn gen ca cc vi sinh vt khc sang cho vi sinh vt v bin t bo vi sinh vt nh nhng nh my sn xut cc cht cn thit cho con ngi.

C th vi sinh vt c c im:

L c th n bo c chu k sinh trng ngn ( Trao i cht mnh.

D ng ho nhiu loi nguyn liu khc nhau.

Sn xut cng nghip hng lot v c kim sot.

2.2.2.1. Phng php to dng

1) Cc bc c bn to dng vi sinh vt

Chun b DNA mc tiu: DNA mc tiu c khuch i trc tip t DNA b gen thng qua PCR hay ct t mt lng ln DNA b gen ban u.

Chn vect ph hp vi t bo ch, mc ch to dng, kch thc gen ch. Vector phi c x l thch hp.

Chn vector ti t hp: Ni DNA mc tiu vo vector to vector ti t hp.

Bin np: Chuyn vector ti t hp vo t bo ch.

Tuyn chn dng t bo ti t hp: Sng lc v la chn dng TB ch cha phn t DNA ti t hp.

2) Thit k thc nghim

3) Tch dng invitro

L tch dng thc nghim: tch dng gen c thc hin t cc mu hoc tng hp nhn to on gen cn tch dng. Tch dng thc nghim gm cc bc:

Chun b gen cn tch dng.

Chn vector cn tch dng.

To vector ti t hp.

Bin np vector ti t hp vo t bo ch.

Sng lc cc dng ti t hp.

Nui cy dng ti t hp thu sinh khi v protein ti t hp.

4) Tch dng insilico

Tch dng insilico tch dng o: tng hp, phn tch cc kt qu tch dng gen invitro.

C s thng tin t ngn hng d liu gen.

Gip tm kim, la chn nhanh gen cn thit, vector tch dng, xc nh cc im ct gii hn, th nghim m hnh hot ng ca gen, nh gi hiu qu chuyn gen.

a. Nguyn tc

La chn phn mm ph hp: BLAST, pDRAW32, FASTA 3

Thc hin yu cu nghin cu theo mc ch ra

Chn vector tch dng.

Chn DNA insert.

Chn RE thch hp.

Bin np vector ti t hp vo t bo ch.

Th nghim kh nng biu hin gen.

Kt qu tch dng insilico cho php la chn

Phng n thit k vector ti t hp.

D on trc kt qu tch dng.

D on hiu qu chuyn gen.

( d on mc thnh cng ca thc nghim.

Cc bc c bn:

S dng phn mm

Np thng tin d liu

La chn vector tch dng ( tm v tr enzyme ct gii hn

Tu theo mc ch

5) Thu nhn acid nucleic (phn ch mu xanh c thm)a) Ph v vt liu ban u

Vt liu ban u: virus, vi khun, t bo thc vt, t bo ng vt.

PP dng m t bo cng nh nhng cng tt.

PP ti u: phn hy thnh t bo bng enzyme v dng ha cht lm tan t bo, v d nghin m t bo trong hn hp cht ty: SDS, sarcosyl v proteinase, i vi TBTV dng cc phng php mnh hn nh dng my nghin ph v t bo.

b) Kh protein

Nguyn l: protein khi b bin tnh s ko ha tan trong nc c cha acid nucleic.

S dng phenol hay hn hp phenol/chloroform lm bin tnh protein.

Mu c lc tht mnh trong phenol hay hn hp phenol/chloroform.

c) Ly tm tch pha

Pha nc cha acid nucleic.

Pha gia cha protein b bin tnh.

Pha phenol/chloroform.

d) Ta acid nucleic

Mc ch: Thu nhn acid nucleic di dng c c nhm bo v chng khi b tiu hu bi enzyme, c th ho tan li chng trong dung dch theo nng mong mun.

Hai cht kt ta thng dng l isopropanol v ethanol (khng cn s hin din ca mui)

Thu ch phm DNA: dng enzyme ribonuclease lm tan RNA trong ch phm.

Thu ch phm RNA

Cc ch : phn t RNA ko bn, d b phn hy bi enzyme RNase.

RNase c mt khp ni, c rt nhiu trn ngn tay, chng c hot tnh cao v bn vng vi cc tc nhn thng dng loi b enzyme.

Trnh mi tp nhim bi RNase.

Bo qun: - 700C trong cc ch phm RNasine c ch Rnase.

Thu ch phm mRNA

Tinh sch mRNA tng hp cDNA: c th tinh sch tip mRNA bng oligo (dT) cellulose.

Oligo (dT) cellulose bm vo ui poly A. Nh vy s loi b DNA, tRNA, rRNA.

Tch mRNA ra khi mu bng sc k i lc trn ct oligo (dT) cellulose.

Trn th trng c b kit s dng cc vin bi t c mang oligo (dT) trn b mt, mRNA bm ln mt vin bi t.

Thu nhn bi t qua ly tm.6) Cc phng php tinh sch DNA, RNA

a) Siu ly tm

Siu ly tm trn mt gradient lin tc cesium chloride (CsCl) (tinh sch plasmid & phage)

CsCl m c trong ng ly tm s hnh thnh gradient t trng vi t trng tng dn t ming ng xung y ng.

phn gii ca mt gradient t trng c th t 1% n v t trng.

Cc acid nucleic di chuyn trong qua trnh siu ly tm n v tr c t trng bng vi t trng ca chng s ngng li v hnh thnh nn mt lp c nh trong ng.

Thu nhn sau ly tm.

Ly tm ng t trng

Siu ly tm trn mt gradient ko lin tc m chloride (ng dng tinh sch mt lng ln phage)

ng ly tm bao gm nhiu lp m c t trng tng dn t ming n y ng.

Acid nucleic c t trng bit trc.

Acid nucleic cn tinh sch nm mt phn cch hai lp m.

Trong qu trnh ly tm, ch nhng phn t c t trng cao hn t trng lp m th di chuyn xuyn qua lp m.

b)Sc k

Sc k i lc dng tinh sch mRNA (poly U sepharose hay oligo dT cellulose)

Sc k lc gel dng phn tch cc acid nucleic v cc Nu t do sau qu trnh to mu d nh du.

Sc k trao i ion trn vi ct thu hi lng DNA rt nh.

Sc k lng hiu sut cao c dng tinh sch cc oligonucleotide tng hp, plasmid, phn tch cc on DNA.

c) in di

K thut quan trng trong KT di truyn v trc tip lm cho cc on acid nucleic hin th.

Nguyn l: c tnh ca acid nucleic l pH trung tnh chng mang in tch m (nh cc nhm phosphat nm trn khung) nn khi c trong in trng chng s di chuyn v pha cc dng, tc di chuyn nhanh hay chm ca acid nucleic ph thuc vo kch thc ca chng, nng gel v cng dng in.

Thu nhn mu sau khi in di

Ct agar cha cc vch DNA cn thu nhn

Ti lp in trng thu nhn DNA trong ging nh khot trong gel ngay sau vch DNA.

Thc hin trong gel c bit c im nng chy thp.

e) Quang ph k

C s: mc hp thu nh sng khc nhau ca cc base nito trong cc mch DNA, RNA n, kp.

Nng ca dd acid nucleic c xc nh bng cch o hp thu ti bc sng 260 nm trong my quang ph k.

Kim tra tinh sch ca dung dch: do OD 280 nm (bc sng hp thu ca protein), dd sch khi OD260nm/OD280nm = 1,8 2.

Xc nh s lng acid nucleic bng quang ph k

Mi tng quan: mt n v OD260nm tng ng vi

50 (g/ml dd DNA si i.

37 (g/ml cho dd DNA si n.

40 (g/ml cho dd RNA si n.

CDNA ((g/ml) = OD260nm x 50 x (nghch o pha long)

CDNA xc nh chnh xc khi dch chit DNA tinh sch.

BI 2: CC ENZYME THNG DNG TRONG K THUT DI TRUYN2.1. Enzyme gii hn (RE) (c khong 2582 RE)

c tnh c bn: RE kh nng nhn bit v ct mt trnh t xc nh trn phn t DNA ti v tr c bit ( cha kha nghin cu k thut DNA ti t hp.

Ngun gc pht hin:

Khi virus xm nhp vo vi khun theo hai chu trnh tan v tim tan, DNA virus s b h thng bo v ca vi khun tiu dit khi xm nhp vo.

Kho st DNA ca cc hin tng trn, ngi ta pht hin ra h thng R M (Restriction Modification) bao gm hai tnh cht gii hn v ci bin.

H thng R M c hai hot ng chnh:

RE rt chuyn tnh ti mt v tr xc nh, c tc dng tiu ha DNA ngoi sinh.

Modification methylase c nhim v ci tin v bo v DNA ngoi sinh.

Tn Gi

Ch ci u in hoa: ch tn chi ca SV tch chit RE.

Hai ch ci tip theo in thng: ch loi hay ging.

Ch ci tip theo in hoa (nu c): ch tn chng.

Ch s la m: ch s th t dng SV.

2.2. Cc loi enzym gii hn

Loi 1: Enzyme nhn bit trnh t, tip tc di chuyn n cch 1000 5000 Nu k t v tr nhn bit ri ct ( ct khng chuyn bit v nhy nn ko s dng trong KT di truyn.

Loi 2: E nhn bit trnh t v ct ngay ti v tr ( ct chuyn bit nn c s dng trong KT di truyn.

Loi 3: E nhn bit trnh t v ct DNA v tr cch 20 Nu ( ct ko nhy nn ko c s dng trong KT di truyn.

2.3. RE loi 2

Trnh t nhn bit Trnh t t 4 6 Nu (thng l 6 Nu).

C cu trc palindrom tc l hai mch ca trnh t hon ton ging nhau khi c theo chiu 5 3.

V tr ct ging nhau trn hai mch.

Cc kiu ct

Ct to u bng

Hae III

5-GG CC-3

3-CC GG-3

RE ct DNA ti mt im. Sau khi ct hai u bng ko c kh nng kt hp li vi nhau

Ni u bng dng T4 ligase.

Ct to u sole (u dnh)

V tr ct lch nhau trn hai mch.

Cc u so le c th bt cp tr li

Dng ligase thng thng ni.

2.4. Quy trnh thit lp phn ng thy gii bi RE Mi RE hot ng ti u trong iu kin phn ng xc nh, c bit l dd m (theo bo co ca hng sn xut).

RE c bo qun trong dd m cha 50% glycerol - 200C.

Nn tin hnh phn ng trong mt th tch nh, thun li cho s tip xc gia E c cht.

Ch th tch ca RE ko vt qu 1/10 th tch phn ng trnh glycerol c ch enzym.

Trn 1 lng RE vi DNA ch trong dung dch m.

Phn ct ng din ra 370C.

Thit lp phn ng cn nhiu RE

Nu chng hot ng trong cng mt dung dch m th c th s dng c.

Nu chng khc nhau v dung dch m th cho RE hot ng trong dung dch m c nng mui thp trc, sau thm NaCl t nng cao hn cn cho RE kia hot ng.

Ch : RE ko mang tnh c hiu loi do RE c tch chit t VK c th nhn bit v ct cc phn t DNA ca ng vt, thc vt.

Mt s yu t nh hng n phn ng ct

Dung dch m cho phn ng ct.

Nhit .

Trnh t nhn bit b methyl ha.

2.5. ng dng ca RE

Lp bn ct gii hn v to dng biu hin gen.

Lp bng ct gii hn Cn bit v tr ct tng i ca cc im nhn bit i vi E gii hn trn mt on DNA.

Lp bng cc im gii hn.

Phng php

S dng 2 RE tr ln ct on DNA bng mt s RE la chn (ct ring l v kt hp).

Chy in di cc on phn ct.

Xc nh kch thc on phn ct

Xc nh v tr cc im ct ( lp bn RE

BI TP ( Quan Trng)

2.6. Nuclease Nhm E phn ct DNA v RNA mt cch ko chuyn bit ( RE l nhng nuclease nhng mang tnh chuyn bit cao hon).

Nuclease phn hy acid nucleic bng cch lm t mi lin kt phosphodiester ni cc Nu.

Cc loi chnh s dng trong k thut di truyn

DNase I: ct DNA c si n v si kp ti cc im ngu nhin.

Nuclease S1: ct c hiu i vi si n ca c DNA v RNA.

Exonuclease III: to ra cc phn t mang u 5 nh ra.

RNase A: ct lin kt phosphodiester nm ngay sau mt pyrimidin ca RNA mch n.

RNase H: loi b RNA trong mt phn t lai RNA, DNA (loi b RNA trong phn ng phin m ngc).

2.7. Polymerase (DNA v RNA) DNA pol I

ng vai tr quan trng trong hot ng t ti bn v c ch sa sai DNA trong sao chp VK

C hot tnh polymerase (tng hp) v exonuclease (sa sai).

ng dng

Xc nh trnh t DNA.

ng dng trong KT cloning

Tng hp cc mu d c phng x cao.

Xy dng cc vect t DNA mch n.

Enzyme Klenow

Sn phm c hnh thnh t s thy phn nh DNA pol I.

Gi hot tnh Pol v exonuclease 3 5, mt hot tnh exo 5 3 khng mong mun.

ng dng

Xc nh trnh t DNA.

Tng hp cDNA t mRNA.

Gn thm cc Nu vo u 3-OH ca DNA

S l u dnh to u bng.

T4 DNA pol

C ngun gc t phage T4 c tnh cht monomer.

Hot tnh

Polymerase v exo 3 5.

Mt hot tnh exo 5 3 ko mong mun.

Hot tnh exo 3 5 mnh hon 15 ln.

Taq pol

L DNA pol chu nhit, c tch chit t vi khun chu nhit thermus aquaticus.

Khng b ph hy nhit bin tnh, xc tc tng hp t u n cui qu trnh phn ng PCR.

Enzyme phin m ngc ( Reverse transcriptase)

Ngun gc: do cc retrovirus sn sinh nhm sao chp RNA ca retrovirus.

Vai tr:tng hp mt mch DNA b sung (cDNA) t khun RNA theo 5 3 khi c mt primer.

ng dng

Thit lp ngn hng cDNA.

Tin hnh phn ng PCR trn mRNA.

Xc nh trnh t ca nucleic acid.

Terminal transferase

Ngun gc: ly trch t tuyn c ca b. Vai tr: xc tc phn ng gn cc nucleotide vo u 3-OH t do ca phn t DNA (ging klenow) ng dng Thm ui to u so le ca phn t DNA dng trong to dng.

nh du cc u 3-OH ca DNA.

RNA pol Xc tc tng hp RNA t mt mch khun DNA si i theo hng 53. RNA pol I tng hp rRNA, pol II tng hp mRNA, pol III tng hp tRNA. SP6 RNA pol c ngun gc t phage xm nhim Salmonella typhimurium (gy bnh thng hn). T3 v T7 RNA pol c ngun gc t phage xm nhim ecoli. ng dng Tng hp mu d RNA.

Xc nh trnh t ca mt DNA c dng ha.

Nghin cu bn phin m RNA ca mt DNA c dng ha.

2.8. Ligase (keo phn t) ng dng

Xc tc phn ng ni hai u ca hai trnh t DNA hay RNA thng dng trong k thut to dng.

Sa cha mi lin kt phosphodiester b t, gy, h.

Ni cc Nu thng qua u 3-OH v 5-P lin k do c mt im ct.

C ch

Ligase b adenyl ha bi ATP.

Sau ligase s adenyl ha nhm phosphat u 5 ti im ct.

Hnh thnh mi lin ht phosphodiester.

Mt s ligase thng dng

E.coli DNA ligase: xc tc phn ng ni hai trnh t DNA c u so le.

T4 DNA ligase: Ligase c a chung nht trong k thut to dng v c kh nng ni c u so le v u bng.

T4 RNA ligase (ly trch t phage T4 xm nhim Ecoli): xc tc ni hai trnh t RNA bng cch hnh thnh lin kt phosphodiester.

2.9. Enzym sa u

ng dng

C vai tr quan trng trong cng ngh DNA ti t hp, nht l k thut DNA sequencing.

Vai tr trong vic ni li hai on DNA hoc to ra nhng vector c tnh cht th hin.

T4 polynucleotide kinase

Xc tc s chuyn nhm gama phosphat ca ATP cho u 5 ca DNA hay RNA.

Phosphoryl ha cc trnh t DNA ko c nhm phosphat u 5 chun b cho phn ng ligation trong to dng.

nh du phng x u 5 ca DNA.

Alkaline phosphatase

Xc tc s loi b nhm 5-P ca DNA, RNA v cc Nu t do.

Loi b nhm 5-P trn cc trnh t DNA t do.

T4 polynucleotide kinase v Alkaline phosphatase thng c s dng ng thi trn vector v on DNA cn gn xen trong k thut to dng (s dng Alkaline trc T4 pol kinase sau)BI 3: POLYMERASE CHAIN REACTION (PCR)

3.1. nh ngha

PCR l phn ng tng ng tng hp dy chuyn nh E. polymerase nhm khuch i mt on DNA m khng cn s dng VSV sng.

3.2. Nguyn tc

C hai mi chuyn bit (on DNA ngn, c kh nng bt cp b sung vi mt u ca mch khun)

DNA polymerase: ni di, hnh thnh mch mi

iu kin: phi c thng tin v trnh t DNA to mi b sung chuyn bit.

Nguyn tc: Mt chu k gm 3 bc:

Bin tnh: (95eq \o(\a\ac\vs2(, )) C, 2s): tch ri hai mch.

Lai: Cp mi chuyn bit cho mt trnh t DNA (40eq \o(\a\ac\vs2(, ))C - 70eq \o(\a\ac\vs2(, )) C, 1 pht). Xc nh cho bt cp vi khun.

Ko di : (72eq \o(\a\ac\vs2(, )) C, vi pht): DNA polymerase tng hp mch mi.

(Chu k lp li nhiu ln)

3.3. Cch tin hnh PCR

Vt liu khi u (gen mc tiu)

Mi

DNA polymerase: Taq polymerase chu nhit nhng thiu hot tnh c, kim tra.

Hn hp dNTP

Dung dch m: PCR buffer + MgCleq \o(\a\ac\vs0( ,)) (1, 2 chu k ban u s tng hp ko di lun qua khi on DNA mc tiu ( khi in di c th b nhiu)

Qua chu k 3 ( xut hin chnh xc 2 on gen mc tiu (khng b nhiu)

Thng 30 chu k, ti a 40 chu k. Nu mi khng c hiu s to ra nhng on khng mong mun.

3.4. Cc yu t nh hng n phn ng PCR

Mi: (dng Tin-Sinh: Thit k mi ti u)

Enzyme

DNA mu

dNTP

Nng MgCleq \o(\a\ac\vs0( ,))

3.5. Xc nh ch nhit

Nhit bin tnh

Nhit gn mi: mi bm vo khun (quan trng nht)

+ teq \l(\o\ac(0, )) qu cao: khng bt cp.

+ teq \l(\o\ac(0, )) qu thp: cc th lai khng tng xng rt bn vng, c nhiu cp base khng b tr cho nhau.

Ko di: tng hp DNA

Xc nh nhit nng chy Teq \l(\o\ac( ,m)) ca cp lai khun v on mi bng thc nghim.

Teq \l(\o\ac(0, )) gn mi l tng nh hn Teq \l(\o\ac( ,m)) t 1eq \l(\o\ac(0, ))C - 2eq \l(\o\ac(0, ))C.

Thit k 2 on mi c cng teq \l(\o\ac(0, )) gn mi.

Kim chng teq \l(\o\ac(0, )) nng chy thc nghim Teq \l(\o\ac( ,m)) ca cp lai khun v on mi.

Cng thc tnh Teq \l(\o\ac( ,m)):

Teq \l(\o\ac( ,m)) = (4x[G + C]) + (2x[A + T])C

3.6. MI

3.6.1. nh ngha: l nhng on DNA nhn to ngn (50 bp) gn chnh xc vo v tr ban u v kt thc ca phn t DNA (ch gn 2 u).

3.6.2. c im

Tnh c hiu

Tnh n nh

Tnh tng thch

3.6.3. Thit k mi ti u

Trnh t mi l cha kho PCR

Mi qu ngn: c kh nng bt cp vi cc on khng mong mun (bt cp khng c hiu)

Mi qu di: nh hng n bt cp mi v khun DNA. S bt cp hon ton vi phn t khun khng kp xy ra trong thi gian chu k phn ng ( D b li phn ng.

Tiu chun c bn ca mi: Chiu di: 18 - 30 base

Thnh phn cc base: 45 - 55% (G + C)

Teq \l(\o\ac(0, )) nng chy: 55 - 80eq \l(\o\ac(0, ))C

Trnh t 3 kt thc phi khp vi on DNA ch.

on cui 5 ca mi c th khng cn b sung vi DNA

Teq \l(\o\ac(0, )) bin tnh ca mi phi ging nhau.

Nhng vn khng mong mun

Cu trc kp tc: Khi on mi t bt cp vi chnh n. Cu trc kp tc cn c th t ko di do DNA polymerase lm bin i cu trc mi v mt kh nng gn vi trnh t ch. S nh trng: 2 mi bt cp vi nhau.

3.6.3.1. CHNG TRNH THIT K V PHN TCH MI

1) Chy chng trnh:

Nhp trnh t DNA cn thit k mi

Ci cc thng s: chiu di, nng G, C.

Chy t ng ( kt qu.

2) Phn tch mi: Nu khng s dng cc phn mm thit k.

3) Cc yu t nh hng:

DNA mu

tinh sch

S lng

di

Enzyme: Taq DNA polymerase tch chit t VK chu nhit

(Web: oligo.net hocGenome.www2.stanford.edu/cgibin/SGD/webprinmer)4) Phn tch kt qu

Chy in di: in di trn gel Agarose hoc polyacrylamide.

Phn tch trnh t trc tip ca sn phm PCR: gii trnh t.

Cc phn tch lin quan n nhn dng (Ti t hp sn phm PCR vo vector).

Nu xy ra kch thc sai xt m khng pht hin c ( s dng my gii trnh t (nhn ra s khc bit 1 vi nu)

Cch khc: To mi c gn trnh t RE ( sn phm PCR ( c thm 2 u di hn DNA khuch i ( u sole ( gn vo vector (Ti t hp)

3.7. Gi tr s dng PCR: Thi gian nhanh

n gin, t tn km.

tinh sch ca mu khng cn cao.

3.8. Hn ch:

Phi bit trnh t Nu ca on cn khuch i.

Taq polymerase: (cng v sau cng c s ci bin) t sa sai.

Ngoi nhim.3.9. MT S PHNG PHP PCR THNG DNG

3.9.1. PCR t (Nested PCR)

Nguyn tc: Sn phm PCR ln mt khuch i ln na mt on nm bn trong on vi cp primer khc nm bn trong on DNA ch.

u im: tng nhy, c hiu PCR v s dng cho DNA di.

Nhc im: D ly nhim, i hi kin thc v trnh t DNA ch.

V d: Trong phn ng pht hin virus HDV gy ung th c t cung, ngi ta dng cp mi MYO/MY11 khuch i 1 on DNA 450 bp vng L1 ca HPV, sau khuch i tip on nh hn l 150 bp nm trong on gen trn vi cp mi GP5+/GP6+

3.9.2. Multiplex PCR

L phn ng cng mt lc khuch i c lp nhiu on DNA ch trong cng mt ng nghim.

u im: Tit kim thi gian, h gi thnh xt nghim

Nhc im: S dng nhiu primer cng mt lc lm tng nhng vt ph khi chy trn gel

3.9.3. nh lng PCR bng REAL-TIME PCR

L phng php gip theo di s tin trin ca phn ng PCR theo tng chu k

Vic nh gi xy ra trong sut qu trnh ca phn ng PCR cho n khi kt thc

Lng sn phm PCR c phn ra ti mi chu k nh tn hiu pht hunh quang vi nguyn tc s lng bn copy tng ln dn n s gia tng tn hiu hunh quang.

(Dng ng chun th hin mi tng quan s lng VSV v pht hunh quang

Cc cht pht hunh quang thng dng:

a) SYBR Green I:

Khi thm SYBR Green I vo mu th, n lp tc gn vo DNA si i hin din trong mu th.

Cng nhiu sn phm PCR si i th cng flourescence cng cao v t l vi lng sn phm PCR to thnh. Nhc im: To dng tnh gi

b) on d Taqman:

Nguyn tc:

on d l 1 oligonucleoticle si n bt cp vi 1 si n DNA ch.

on d c hai u: u pht hunh quang F v u c ch Q Khi bt u chu k, si DNA i din c tng hp ng n u F ca on d v bt cp F (F c t do thot khi s c ch ca u Q nn pht hunh quangc) on d Beacon

C cu trc trm ci, bao gm 1 si nucleoticle v mt cung cha G v C gi vng cu trc trm ci.

Mt u F ca on d gn vi cht pht hunh quang v u Q gn vi cht c ch Khi 2 u ny gn nhau th s pht hunh quang khng xy rad) on d lai:

Phng php ny s dng 2 on d c thit k lin tc

u 5 ca 1 on c gn tn hiu pht hunh quang (F1) u 3 ca on cn li cng c gn tn hiu pht hunh quang (F2) Khi c hai on d c gn vo si DNA ch dng si n, 2 u F1 v F2 cnh nhau, nng lng t F1 truyn sang F2 gip F2 pht hunh quang Phn tch kt qu PCR:

- in di

- Phn tch trnh t trc tip ca sn phm PCR

- Cc phn tch lin quan n nhn dng sn phm PCR3.10. XC NH TRNH T ACID NUCLEIC

3.10.1. Phng php ha hc Maxam & Gilbert:

Bc 1: Cc phn t DNA c nh du bng 32P 1 u (5)

Bc 2: Phn ng ct chuyn bit:

X l methy ha lm mch t G(1) PH=2 lm t mch A hoc G(2) Hydrazin: lm t mch C hoc T(3) Hydrazin nng mui cao: Lm t mch C(4)

Bc 3: Sau khi x l, mi nhm cha mt s on nucleoticle c chiu di khc nhau;

a 4 nhm ln 4 bn gel accrylamide pha cc m

on ngn hn s di chuyn xa hn v pha cc dng

Kt qu: Kt qu in di (Xp cc on DNA theo th t t ngn n di (C mc chun l u 5 mang 32P)

V d: 3.10.2. Phng php Dideoxy ca Sanger

Nguyn tc: Khi mch DNA ang c tng hp nu gp (dideoxynucleotide) th b dng li v cc on nucleotide c di khc nhau c to ra.

Dideoxynucleotide l nhng eoxynucleotide trong nhm 3OH c thay bng H

Cc dideoxynucleotide khng cn kh nng hnh thnh cc ni phosphodiester dn n ngng qu trnh tng hp.V d:3.10.3. My xc nh trnh t Acid nucleic:

Da vo nguyn tc Sanger c ci bin

Mi loi dideoxynucleotide c nh du bng 1 fluochrome c mu khc nhau.

Tt c cc dideoxynucleotide cng chm dt ti 1 loi dideoxynucleotide s c cng 1 mu

in ly trn gel polyacrylamide

Kt qu c qua h thng my VT

Cc thng s v trnh t c so snh v phn tch trn my vi tnh: Dch sang trnh t a.a

Xc nh cc im ct ca enzyme gii hn

Cc on tng ng

Cc cu trc khc: promoter, cc on c chc nng iu khin

PCR:

Phi hp phng php PCR v s dng cc dideoxynucleotide

Gii hn:

+ Vng cn xc nh trnh t bit trc

+ Nhm pht hin nhanh t bin im

Phng php:

+ Khuch i on DNA cn xc nh trnh t bng PCR

+ Tch ri 2 mch phn t DNA

+ Bt cp vi mi

+ Phn ng tng hp tng t nh phng php dideoxynucleotide

BI 4: PHNG PHP TO DNG4.1. Cc bc c bn

1) Chn vector: Xt cc yu t: kch thc on DNA l v mc ch to dng2) X l vector: ct bng RE v x l khng b chp li 3) X l DNA l: x l DNA tng ng vi vertor v x l hai u DNA l4) Vector TTH: Trn chung vector, DNA l v ligase. Tinh sch vector TTH (tch chit v ta)5) Chuyn vector TTH vo TB ch. S dng b my ca TB ch to dng vector TTH6) Pht hin dng cn tm

4.2. Vector nhn dng

4.2.1. Cc c tnh c bn

1. C kh nng t sao chp trong TB ch2. Khng ph thuc s sao chp b gen TB ch3. Tn ti trong TB ch qua nhiu th h v khng gy nh hng mi hot ng ca t bo ch4. Kch thc nh5. C cc v tr nhn bit RE6. Mang nhng c tnh gip d dng pht hin t bo VK c mang vector cha gen chn lc.

4.2.2. Nguyn tc chn vector nhn dng1. Da vo kch thc hay bn cht ca on DNA.2. S tng ng cc v tr c hiu ca cc RE.3. Thch hp vi TB ch.4. C kh nng ti bn trong t bo ch.5. To s lng bn sao ln.6. C gen ch th nhy.7. Vng a ct ni nm trong gen ch th.8. Hiu qu nhn dng cao: t l to thnh vector ti t hp cao.4.3. Vector biu hin

1. L nhng vector nhn dng c mang cc promoter mnh, c kh nng dch m to nhiu protein.2. Vector gi nguyn hot tnh khi ci, gn on DNA c kch thc ln3. Khng nh hng t bo ch4. C nhiu trnh t RE5. Gen ch th d dng nhn bit 6. Thnh phn chnh: promoter mnh, v tr bm ca ribosome, tn hiu kt thc dch m, cc trnh t a ct ni, gen ch th.7. Mang ori mnh gip to ra s lng ln bn sao ca DNA ch trong t bo ch.8. Cc loi vector thng dng: plasmid, phage, cosmid, YAC. La chn vector sao cho ph hp vi kch thc DNA ch v mc ch to dng.4.4. Vector ti t hp4.4.1. Cc phng php ni Cc phng php ni

Ni u bng dng T4 DNA ligase (ni c u bng v u dnh) Ni u dnh dng DNA ligase.

Phng php sa u

Dng cc on polylinker.

Dng enzyme terminal transferase.

Dng mi PCR.

4.4.1.1. Phng php dng cc on ni Tng hp cc on oligonucleotide c trnh t palindrom c hiu vi mt RE.

Dng T4 DNA ligase ni vo on DNA l.

Ct bi cng mt loi RE i vi vector v gen cn chuyn.

Rp v ni lin bng ligase.

Thu DNA ti t hp.

Nhc im: Nu DNA c cha cc im gii hn ca RE s dng ct linker th phi dng methylase bo v tt c cc im nhn dng bn trong khi b phn hy bi enzyme.

4.4.1.2. Phng php dng u lch Ct DNA cn chuyn v vector vi cng loi mt RE.

Vector to thnh dng thng c hai u lch.

DNA cn chuyn to thnh DNA thng c hai u lch tng t.

Trn ln v ni nh ligase.

4.4.1.3. Phng php dng enzyme terminal transferase Terminal transferase gn cng mt loi Nu thnh chui vo u 3-OH ca DNA.

Vector: ct bi RE, b sung poly G v enzyme TT.

DNA l: exonuclease 5 3, b sung poly C v enzyme TT.

Trn vector v DNA l.

Lp ch trng nh DNA pol I v DNA ligase.

Thu vector ti t hp.

4.4.1.4 To dng bng PCR Gn thm vo ui primer nhng on gen c trnh t tng ng vi RE

Nhm trc s dng vector ct bi RE no ( t khi dng c hai trnh t bng 1 RE (tnh nh trng), khng cn x l 2 u bng vector.

BI 5: THAO TC BIU HIN GEN PROCARYOTE5.1. Pht hin dng cn tm

Sau bin np, thu c 3 loi t bo:

TBVK ko nhn c plasmid.

TBVK nhn c plasmid nhng ko c gen l.

TBVK nhn c ng plasmid ti t hp.

Cc phng php pht hin

S dng cc oligo tng hp

S dng khng th.

Lai acid nucleic.

Mt hot tnh do xen on.

Pht hin kiu hnh nh gen Lac Z

1. S dng cc oligo tng hp Tch chit v tinh sch protein.

Vi trnh t ngn (15 20 acid amin) ca protein c xc nh.

Xc nh cc trnh t kh hu cng tng ng vi trnh t acid amin.

Xc nh trnh t no ph hp nht vi t bo bng phn mm m phng.

Tng hp cc mu d oligo.

Lai mu d bng PP lai ti ch.

Nhc im Mt nhiu thi gian.

Sai xt ln.

Phc tp trong la chn on oligo thch hp.

2. S dng khng th iu kin: cc khng th c trng cho protein m ha bi gen cn to dng, vector cha cc yu t cn thit cho s dch m.

Thc hin

a phn gii trn b mt thch ph y vi khun, t kch thc t 1 2 mm.

t mng nitrocellulose c tm IPTG cht cm ng ca operon Lac.

Protein hp th ln b mt mng.

mng lai vi khng th c trng.

Phc hp khng th - protein c pht hin nh protein nh du bng 125I c kh nng gn vi khng th.

Pht hin bng phng x t ghi cc a phn gii tng ng vi tn hiu c thu nhn li.

Nhc im Phi s dng vector biu hin (ko s dng vi vector nhn dng).

3. Lai acid nucleic S dng mu d:

Lm tan ti ch trn giy lc nitrocellulose

DNA thot ra gn trn giy lc

Lai vi mu d

Pht hin Nhc im: Phng php ny ko tn dng c cc c tnh ca vector

Cc bc tin hnh phn trn.

4. Mt hot tnh do xen on vi vector c mang hn 2 gen khng thuc

5. Pht hin kiu hnh nh gen Lac Z

Gen Lac Z l gen ch th c gi tr cao, d nhn bit dng ti t hp nh mu sc ca khun lc.

C ch

Gen Lac Z m ha cho enzyme ( - galactosidase khi c cht cm ng IPTG (Isopropyl-B-D-thiogalactopyranoside)

Khi c mt X gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside), ( - galactosidase s thy phn X gal t mt cht ko mu thnh mt cht c mu xanh.

Kt qu pht hin

Nu khun lc thu c c mu xanh ( ko c dng ti t hp do on DNA ch ko c chn vo gen Lac Z.

Nu khun lc thu c c mu trng ( thu c dng ti t hp do on DNA ch c chn vo gen Lac Z lm cho gen Lac Z ko hot ng.5.2. Biu hin ca gen c to dng

Mun gen to dng c biu hin protein th vector phi c y cc yu t phin m v dch m - vector biu hin

Phin m: gen nm gia promoter v du kt thc

Dch m: mRNA cn phi mang pha u gen trnh t rbs-im bm ribosome

5.2.1. Biu hin gen nhn dng

Chn mt gen vo 1 vect. Vy: Lm sao m bo s biu hin thnh cng? Nht nh s biu hin?

Cng tng hp cao protein ti t hp.

Cc vn pht sinh.

Thit k promoter nh th no?

Phin m v dch m?

r - protein khng bn?

Tnh n nh? Vect ti t hp d b bin mt.

Oxy sinh trng khng ?

Gnh nng trao i cht?

( Th no l mt h thng biu hin l tng?

5.2.2. Cc gii php5.2.2.1. Promoter

H thng biu hin gen cn phi c trnh t promoter mnh iu chnh, nm trc gen nhn dng. (Yu cu ti thiu cho 1 h thng biu hin Gen)

Promoter mnh: c tnh i lc cao vi RNA polymerase ( vng nm sau thng xuyn phin m.

Promoter iu chnh: kim sot mc phin m chnh xc.

( Vy promoter mnh c ti u khng?

Khi chn gen mc tiu vo vect di s kim sot ca promoter mnh, phin m lin tc.

Mc cao, biu hin lin tc.

( T bo ch kit qu nng lng ( suy yu chc nng cn thit t bo ch ( t bo ch mang gen nhn dng sm b ln t bi cc t bo khng c vector ( Khng phi l gii php ti u

( Gii php: promoter mnh nhng phi iu khin c.

Khc phc:

Ch biu hin giai on c bit trong chu k sinh trng ca t bo ch.

Ch biu hin trong 1 khong thi gian xc nh.

( Promoter iu khin: to cho t bo c th kim sot mc phin m chnh xc.

Vector biu hin.

5.2.2.1.1. Cc h thng promoter mnh.

Operon Lac

Operon Trp

Promoter Tac: Bn thit k in vi tro vng -10 ca Lac, vng -35 ca Trp.

peq \l(\o\ac(L, )): pro ca phage Lamda.

Promoter ca phage T())eq \l(\o\ac( ,)).

Ti sao promoter mnh?

Pro tng tc vi cc th c ch ( th c ch l cng tc kim sot qu trnh phin m tc l iu khin c.

iu khin pro mnh nh th no?

Holoenzyme RNA polymerase nhn bit promoter.

Holoenzyme c to ra khi: Yu t Sigma kt hp vi RNA polymerase li. Yu t Sigma hng ln

a) Promoter peq \l(\o\ac(L, )).

Promoter ca phage Lamda.

Kim sot bi protein c ch CI ca phage Lamda.

cI (CI.857) iu ho s phin m peq \l(\o\ac(L, )).

Protein c ch (cng tc) - cI iu ho nh th no?

cI nhy vi nhit : bt hot 42eq \l(\o\ac(0, ))C

Cc t bo mang cI pht trin 28 - 30eq \l(\o\ac(0, ))C. cI ngn chn qu trnh phin m peq \l(\o\ac(L, )).

( Cng tc cI: Khi mun cy t bo t giai on mong mun, nng nhit bng 42eq \l(\o\ac(0, ))C ( cI bt hot ( phin m c thc hin.

Th c ch l 1 cng tc qu trnh phin m ca cc promoter v:

Qu nhiu phn t c ch: Kh cm ng phin m.

Qu t phn t c ch

T l gia cc phn t c ch trn s bn sao ca trnh t promoter?

Gii php 1: Gen protein c ch v promoter tng xng ca n t trong 2 plasmid khc nhau ( duy tr s bn sao khc nhau cho mi t bo ( duy tr t l thch hp.

Vd: Gen c ch t plasmid c s bn sao thp (1 - 8 bn), gen nhn dng c 30 - 100 bn.

Gii php 2: (Vector biu hin dch m)

ti u hiu sut thu hi sn phm gen:

Pro mnh v iu khin uc

Hiu qu dch m.

bn ca r-protein.

C s phn t:

S c mt ca tn hiu khii ng dch m.

Vng lin kt vi ribosome trong mRNA phin m.

b)Operon lac ( S hin din ca lactose ( iu khin cht km hm (lm thay i cu hnh ca cht km hm ) ( n khng gn vo gen lm chng ngi vt gnh nng TC ( lm chm qu trnh sinh trng.

c)Operon Try: cht km hm (Ngc li so vi Operon Lac)

Mi trng khng c Try ( cht km hm khng gn vo Operator ( s thc hin qu trnh phin m.

Mi trng c Try (cht ng km hm) ( cht km hm gn vo operator ( phin m khng th thc hin c.

BI 5: VECT BIU HIN DCH M

Vng lin kt vi Ribosome trong mRNA phin m. Trnh t 6 - 8 nu.

V d: UAAGGAGG (trn mRNA) bt cp.

AUU.. (trn thnh phn RNA ca tiu n v nh Ribosome)

Lin kt mRNA v rRNA cng mnh ( hiu qu

Nhiu vect biu hin E.coli c thit k m bo RNA cha vng lin kt Ribosome mch c th dch m trong E.coli.

6.1. Cc iu kin b tr vng lin kt ribosome Trnh t lin kt ribosome phi t cch Codon khi u dch m 1 khong chnh xc.

Trnh t DNA s cha vng lin kt Ribosome.

Vi Codon u tin ca gen mc tiu khng cha cc trnh t m sau phin m c th b cun li ( Vng lin kt ribosome phi t ng v tr v cu trc bc 2 ca mRNA khng cn tr s tip xc ribosome. (Cu trc bc 2 ca mRNA: xon cun, bnh thng mRNA khng thng).

Vect biu hin ( H thng vect thng dng kt hp c tn hiu phin m v dch m ( ch cn s dng cng tc iu khin trong phin m).

6.2. H thng vector thng dng Cc h thng vector thng dng thng c:

Gen khng Amp lm du chun ( sng lc).

Promoter Tac

Vng lin kt ribosome LacZ Codon khi ng ribosome ATG cch vng lin kt ribosome theo chiu xui.

Cc yu t kt thc phin m Teq \l(\o\ac( ,1)), Teq \l(\o\ac( ,2)) t phage.

Gen nhn dng chn vo vng RE, nm gia vng lin kt ribosome v cc yu t kt thc phin m.

Sau khi cm ng ( phin m ( Dch m.

6.3. Cc pht sinh khc.

6.3.1. S khng tng thch vi t bo ch.

Gen nhn dng c cc codon rt him khi c t bo ch dng.

V d: AGG, AGA, AVA, CVA, CGA ( E. coli)

T bo ch khng tng hp tRNA ( hiu sut sinh tng hp thp.

( Gii php:

Nu gen mc tiu c ngun gc t Encaryote ( Nn nhn dng v biu hin trong mt t bo ch Encaryote.

Nu phin bn mi ca gen mc tiu cha cc codon m c t bo ch s dng thng xuyn (c th tng hp ho hc).

Mt t bo ch ci bin: Dng E.coli sn xut vt tri cc tRNA ca codon AGG, AGA, AUA, CUA ( thng mi).

6.3.2. S kit qu Oxy

VSV sinh trng v pht trin bnh thng: cung cp oxy lun lun l vn ln t ra trong cng ngh ln men.

i vi chng TTH: oxy cng b gii hn, kit qu.

Thiu oxy ( pha log chm ( Nhanh chng chuyn sang pha n nh ( phn hu r-protein.

( Gii php:

Gii php 1: S dng nhng chng khng c protease.

Khng n gin.

N c chc nng gi nh.

Phn hu cc protein khng bnh thng; sai hng.

Gii php 2: To chng t bin 1 hoc nhiu gen protease.

Khng n gin.

Protease c chc nng gi nh.

Phn hu protein khng bnh thng hoc sai hng.

Gim protease ( t bo sinh trng v pht trin yu hn.

Gii php 3: a gen m ho cho Hemoglobine tng lng oxy ni bo.

Khng n gin nhng kh thi.

Vitreoscilla: Gram (-) ; a kh, sng ao t ni ngho oxy, sinh tng hp 1 phn t tng t Hemoglobine lin kt oxy mi trng ( tng oxy sn c.

Khai thc: Gen m ho hemoglobine c nhn dng v biu hin E.coli (khai thc tnh nng E.coli).

6.2.3. Quy m ln men ln.

Quy m nh: cm ng bng nhit hoc cht cm ng ho hc.

Quy m ln:

Thay i nhit : cn thi gian kh di v nng lng ( gi thnh cao.

Cht cm ng ho hc: cng khng kinh t.

( Gii php: H thng 2 plasmid (s dng promoter peq \l(\o\ac(L, )) ln men dng quy m ln)

Plasmid th nht.

cI c t di s kim sot promoter Trp.

a vo plasmid c s lng bn sao thp.

Khng tng hp d tha cI.

Plasmid th hai.

Trp- (khng c tryptophan): promoter m ra ( tng hp cI ( promoter Peq \o(\a\ac\vs2(, )) ng li.

Trpeq \l(\o\ac(+, )) (trp cht ng km hm): promoter trp ng li ( khng tng hp protein c ch cI ( promoter peq \o(\a\ac\vs2(, )) hot ho m ra.

(Khi no khng cho promoter hot ng ( khng b sung trp ( phi b sung ng lc)

H thng 2 plasmid hot ng nh th no?

Cho cm ng biu hin gen nhn dng bng cch b sung trypton vo mi trng.

H thng 2 plasmid l 1 cng c khng t tin sn xut ra r-protein quy m ln.

6.2.4. Chng ti t hp bin mt.

Nguyn nhn:

Khi tng bn sao ca gen nhn dng ( sinh tng hp protein tng (

Cc t bo khng c plasmid c xu hng pht trin nhanh ( n mt thi gian s chim ht dch nui cy ( chng TTH bin mt (c th c t l nh sng lc).

Gii php:

Gii php 1: B sung cht khng sinh: to kh nng ch c t bo TTH pht trin mnh.

Gii php 2: (c bn) a thng DNA nhn dng trc tip vo DNA NST ca t bo ch.

(thm 1 plasmid l thm gnh nng chuyn ho cho t bo.)

Cc iu kin tin quyt:

V tr ci nhp ca gen mc tiu trn NST khng c nm trn exon m ho thit yu (nh hng trc tip n hot ng ca t bo ch.)

Gen mc tiu phi a vo v tr khng quan trng, khng cn thit.

Vn phi thit k promoter kim sot ( bn)

DNA mc tiu phi c tng ng trnh t vi NST ( trao i cho) (Khong 50 Nu) xy ra TTH hoc trao i cho.

Quy trnh:

Nhn dng v tr ci nhp trn NST

Phn lp, nhn dng vng ci nhp NST.

Thit k: Ni gen mc tiu - promoter - V tr ci nhp.

Chuyn bn thit k vo t bo ch.

Chn lc, duy tr, n nh.

( Din tin xy ra:

DNA trn plasmid bt cp vi trnh t tng ng trn NST ch.

C th xy ra trao i cho (xc tc bi Enzyme)

Ti t hp

( S lm gim thiu gnh nng trao i cht. (Gen nhy cng l mt gii php).

6.2.5. Loi b gen nh du chn lc.

Vai tr gen nh du chn lc.

Tr gip nhn dng t bo bin np (nhng ch trong iu kin phng th nghim).

( Tnh hung: Gen khng khng sinh pht tn ra mi trng ( gy bnh cng ng.

( Gii php: Ch c gen mc tiu c ci nhp vo NST ch v c cc gen nh du chn lc cng c chn.

Thit lp h thng chn - thi (Nu khng VSV TTH khng c a ra th trng)

X l: Gen nh du chn lc chn 2 u bng trnh t RE.

Chn gen nh du chn lc x l vo plasmid.

Gen c th c ct ra khi khng cn thit chn lc.

Cre: enzyme

LoxP: 34 bp.

Gen nh du chn 2 u bi LoxP.

Ci nhp TTH vo DNA NST.

Gen nh du loi bi E.cr.

H THNG CRE - LOXP

E.cre c a vo qu trnh to dng.

Dng cng tc IPTG gii c ch LacI ( tng hp Cre.

Khi xong nhim v: pht trin nhit loi b khi t bo.

( Vic loi b gen khng khng sinh l cn thit m bo ATCNSN chng bin i gen. 6.2.6. Gnh nng trao i cht

Gnh nng trao i cht: - Do thay i trao i cht ca c th

- Suy yu chc nng thng thng.

Nguyn nhn gnh nng trao i cht: Tng s bn sao plasmid, tng kch thc: i hi tng ngun nng lng t bo cho vic sao chp v duy tr plasmid.

Gii hn Oxy: khng cho t bo ch v duy tr biu hin plasmid.

Biu hin cao ca 2 ngun protein nh du v protein ch: Kit qu ngun aminoacyl - RNAt-

Tc nghn v tit xut

Khi protein ngoi lai c biu hin vt mc.

Xut ra khi t bo cht.

Ngn cn s nh v chnh xc cc pro t bo ch cn thit.

Protein ngoi lai: c th xen vo chc nng t bo ch.

Mt s cc t bo ch c bit c tc h hp cao.

H qu ca gnh nng trao i cht.

Gim tc sinh trng ca t bo.

Mt plasmid TTH hoc 1 phn.

Gim c ch c sa sai trong phin m.

C th thay i kch thc v hnh dng t bo ch.

Tng hm lng polysaccaride ngoi bo ( kh khn hn giai on thu nhn v tinh sch.

Gii php:1. S dng vector plasmid c s bn sao thp hn.

2. Gim thiu s dng cc vector plasmid cng nhau.

3. Ci nhp DNA mc tiu vo DNA nhim sc th ca t bo ch.

4. S dng promoter mnh v iu khin c.

- Giai on sinh trng: promoter kim sot dch m ca gen mc tiu c ng li.

- Giai on sau: cm ng promoter m ra.

5. Hy chp nhn s biu hin gen ngoi lai 1 cch khim tn.

Vd: r-protein chim 5% tng s protein t bo nhng mt t bo TTH cao.

Kh t c mc biu hin cao km theo mt t bo cao?

Do s tch t sn phm thi.

c bit l do s tch t acetat, dn n c ch sinh trng ca t bo v sinh tng hp protein.

( Gim tch t acetat l 1 gii php.

Phng n 1: Gim tc ly glucose ca t bo bng cch b sung 1 cht tng t glucose (methyl ( - glucosit)

Phng n 2: Dng k thut di truyn, k thut to dng gim acetat.

* a gen m ho enzyme acetolactat synthase (AS) vo E.coli. AS xc tc s hnh thnh acetolactat t pyruvat, lm gim dng chuyn ho qua acetyl coenzyme A thnh acetat. Do a gen AS a vo E.coli.

* Vi khun dng L cng l 1 gii php. Vi khun dng L: + To thun li cho s bi tit s lng ln cc R-protein vo mi trng.

+ L bin th ca vi khun thng thng, thiu thnh t bo.

+ X l bng cht cm ng (penicillin, lysosyme) Vt peptidoglucan bin mt.

+ Vi khun khng th khi phc c: Vi khun dng L bn v c th s dng trong KTTD.

* Bin np vo vi khun dng L.

- Vi khun dng L c vi DNA plasmid kt hp polyethylen glucol.

- Biu hin: Mc cao - tit hon ton protein ch.

- c trng ca vi khun dng L.

* Nhng hn ch vi khun dng L.

- Khng li nhiu khng sinh thng thng.

- Khng s dng chng nh l cc du chun khng khng sinh trong k thut nhn dng.

- n nh khng cao.

- Pht trin chm hn so vi vi khun thng thng.

- Ch nui cy c bit hn, k thut c bit hn.

( Khc phc:

+ Gen nh du khng m ho cho khng khng sinh.

+ Pht trin h thng ln men c gi tng t nh nui cy t bo ng vt.

* Cc nhn t gip gen biu hin ti a.

1. S lng hp l cc bn sao ca vector plasmid i vi 1 t bo.

2. Chn promoter mnh phin m tt.

3. C trnh t im bm rbs.

4. DNA TTH c s n nh lu di.

5. Trnh s thu gii protein do cc enzyme ca t bo.

* H thng vector biu hin cao.

- Trnh t khi ng promoter.

- Trnh t kt thc.

- Mt cc v tr lin kt ribosome.

- S bn sao ca gen nhn dng.

- Gen c mang trn plasmid hay ci nhp vo b gen ca t bo ch?

- Hiu sut dch m.

- bn ca protein ca gen nhn dng.

- Mc biu hin gen ngoi lai: ph thuc vo sinh vt ch.

Tch chit DNA t b gen ca t bo

Phn lp on gen bng PCR

Vector plasmid nhn dng

Nhn dng trong t bo nhn dng

Tch chit v kim tra trnh t gen

Phn lp gen mc tiu

Vector biu hin

Chuyn vector biu hin c cha gen mc tiu vo t bo nhn dng

Chuyn vector biu hin c cha gen mc tiu vo t bo biu hin

Tch chit v kim tra trnh t gen

Cm ng biu hin protein

Kim nh protein bng SDS PAGE v Western blot

L do dng trong k thut di truyn

i t protein

Thng dng trong VSV