CNG NGH GEN

TOPIC

GVHD: ThS. Nguyn Th Kim Cc NHM : 3 LP: 52CNSH

NI DUNG
TNG QUAN Saccharomyces cerevisiae (???!!!)

II

TO DNG T BO NM MEN BIU HIN -AMYLASE CHU NHIT TRN B MT T BO

I. Tng quan
1. c im nm men L sinh vt nhn chun, c th nui cy vi quy m ln, cu trc gen c gii trnh vo nm 1996 kch thc 1,35107

 C c tnh khng gy bnh, khng to ni c t, c qu trnh ng dng lu di trong thc phm nn S.cerevisiae c xp vo nhm sinh vt an ton GRAS. So vi t bo hu nh, t bo nm men sinh trng tng i nhanh.

 C kh nng to sinh khi ln, thao tc di truyn n gin, protein sn phm c tit ra mi trng nn thu hi d dng. C cc ci bin sau dch m (ng ha, phosphoryl ha) to protein c y hot tnh sinh hc

2. Cc loi vector
Hu ht cc vector s dng trong t bo nm men l nhng vector vn chuyn m n c th sao chp trong c E.coli v S.cerevisiae. Cc loi vector: Vector st nhp YIp Vector YEp (plasmid 2 m) Vector YRp Vector YCp Vector YAC

2 loi vector thng dng

1 2

Vector st nhp YIp

Vector YEp

Vector st nhp YIp

Vector st nhp c tnh bn vng cao, ch mt 1% qua 1 th h, khng cn p lc chn lc.

S st nhp vector vo b gen ca nm men xy ra nh s trao i cho gia vector v nhim sc th to nn 2 dng DNA tng ng mang gia n l trnh t DNA ca vector. Tn s st nhp khong 1-10 th bin

vector YEp (plasmid 2 m)

S. cerevisiae c th cha plasmid t nhin gi l plasmid 2 m, c kch thc 6318bp, s bn sao trong t bo khong 50-100, chim 1-2% tng DNA

Plasmid 2 m c s dng thit k cc vector t sao chp c lp trong t bo nm men l YEp.

Tn s bin np ca plasmid rt cao, t 104 105 th bin np/g DNA, thng rt bn vng, ch b o thi mc 1% trong mt t bo

3. Gen chn lc Cc gen m ha cho cc enzyme cn thit cho qu trnh sinh tng hp cc cht dinh dng thit yu cho t bo. Cc gen chn lc tri c tm thy t gen cc t bo ch nm men hoang di nh CUP1 hoc t nm men bin i di truyn nh G418R .

4. Promoter Cc promoter c nhn bit bi RNA polymerase ca t bo nm men chia lm 2 vng chc nng Nhn t TATA - ng vai tr trong vic xc nh v tr khi u phin m. - S phin m thng thng bt u t nucleotide th 30 sau trnh t TATA. Nhn t iu ha thng lu - Nm thng lu nhn t TATA. - L v tr gn cc protein iu ha

Cng ngh biu hin gen m ha enzyme - amylase chu nhit trn b mt t bo Saccharomyces cerevisiae.

A. Vt liu

1. Cc vi sinh vt

Escherichia coli DH5

Saccharomyces ceereviseae MT8-1

2. cc plasmid

Plasmid pICAS1

Plasmid pKTH10

- C kch thc 6,8 kbp, mang gen khng neomycine v on 2,2kb cha gen amyQ m ha cho enzyme -amylase t B.amyloliquefaciens. - Plasmid pKTH10 ny c cung cp bi gio s ngi c.

Pbluescript

C kch thc 2958bp, cha gen chn lc Amp

3. Thnh phn pn ng PCR

Taq DNA polymerase. Pfu Turbo DNA polymerase. 10X PCR buffer: dng cho Taq polymerase (KCl 500mM, MgCl2 15mM, Tris-HCl 100mM, pH9). 10X Pfu PCR butffer. dNTP.

Cp mi khuch i gen amyQ

4. Enzyme ct gii hn

XhoI NhoI HincII

B. Quy trnh thc hin

Chun b vector v DNA ch

Chun b E.coli kh np (DH5)

pICAS1

Thu nhn pKTH10

PCR vi cp mi c hiu cho amyQ Cp mi ny to v tr ct XhoI v NcoI

Thu nhn

pBluescript
bng pp SDS-kim Thu nhn pICAS1 bng pp SDS-kim

amyQ XhoI NcoI

1. To dng Ecoli pICAS1/DH5, pBluescript/DH5

a. Chun b E.coli kh np
50ul E.coli DH5 500ul dch nui cy Ra sinh khi thu c bng 1ml CaCl2, 100mM, lnh
Ly tm thu sinh khi 13000 vng trong 20 pht, 40C

Lc qua m 370C 5ml LB lng

Lc trong 2-3h 370C 5ml LB lng

Ly tm thu sinh khi 13000v trong 20 pht, 40C

Bo qun bng glycerol 80%, - 800C

Thc hin bc 1 v 2 ba ln

Huyn ph sinh khi trong 1ml CaCl2, trong t nht 1h 2

b. Bin np plasmid vo E.coli DH5

1ul plasmid 50ul dd CM 1X 50ul tb E.coli DH5a kh np Mt LB 2% agar + Apicillin: 50ug/ml

vo a petri t trong nc 5

Gy sc nhit 42C 42
Tri trn a mt LB c Ampicillin 37C trong 16h

lnh nc 5

Tin hnh ng thi mt mu i chng khng b sung plasmid

c. Kim tra kt qu v chn dng mang plasmid mong mun Sau khi 37C trong 16h, kim tra kt qu bin np bng cch quan st s pht trin ca E.coli trn cc a bin np v i chng. Chn cc khun lc to v ri rc trn a bin np, cy vo 5ml mi trng LB lng c b sung 5ul Amp 50mg/ml. Sau , nui cy lc 37C. Chn dng E.coli DH5a tng trng mnh trn mi trng LB-Amp.

c. Kim tra kt qu v chn dng mang plasmid mong mun

d. Tch chit plasmid t cc dng E.coli bin np theo pp SDS kim

2. Chun b plasmid mang gen ch

Plasmid pKTH10 mang gen amyQ thu c lm khun mu, thc hin phn ng PCR vi cp mi c hiu cho gen amyQ kim tra plasmid thu c sau khi tch chit t cc th E.coli DH5a.

Tnh phn phn ng PCR: .

Mu chng dng s dng plasmid pKTH10 chun. Sn phm PCR c in di trn gel agarose.

3. To dng pBAmyQ/DH5a
a. Chun b DNA gen mc tiu. - Gen amyQ c khuch i t khun mu l pKTH10 bng phn ng PCR. Cp mi c hiu cho gen c thit k to v tr ct cho enzyme ct gii hn l XhoI v NcoI.

on gen c mang cc trnh t ct chuyn bit cho enzyme ct gii hn XhoI v NcoI.

3. To dng pBAmyQ/DH5a
b. Chun b DNA vector dng ha pBluescript

Phn ng ct vector pBluescript bng enzyme ct gii hn HincII. in di. Ct v thu nhn gel c cha vch DNA cn thit. Dng b kit SephaglasTMBand Prep tinh sch mu ct.

Ct bng HincII

3. To dng pBAmyQ/DH5a
c. Chn gen amyQ c vo vector pBluescript bng phn ng ni di tc dng ca ligase. Thnh phn phn ng ni:

3. To dng pBAmyQ/DH5a

3. To dng pBAmyQ/DH5a
d. Bin np v sng lc pBAmyQ/DH5a

DNA dung hp gia gen mc tiu v vector c bin np vo E.coli DH5a.

Chn cc th bin np da vo cc khun lc xanh trng.

Tch chit plasmid.

3. To dng pBAmyQ/DH5a
e. Kt qu bin np

4. To dng pICAmyQ/DH5a
a. Chun b DNA vector biu hin pICAS1 v DNA gen mc tiu.

Vector pICAS1 c mt v tr ct gii hn duy nht ca enzyme XhoI v NcoI vng MCS. Tin hnh m vng bng 2 enzyme trn tin hnh phn ng ni. Vector pBAmyQ c mang gen amyQ cng c ct bng 2 enzyme ct trn. in di trn gel agarose 1% v thu cc on gel c cha cc DNA plasmid pICAS1 m vng v gen amyQ c ct gii hn hai u. Dng b kit SephaglasTMBand Prep tinh sch mu ct.

4. To dng pICAmyQ/DH5a

4. To dng pICAmyQ/DH5a
Kt qu in di

4. To dng pICAmyQ/DH5a
b. Chn gen mc tiu vo vector biu hin pICAS1
on gen amyQ v vector pICAS1 sau khi tinh sch cha 2 u dnh ca enzyme NcoI v XhoI c ni bng phn ng ni di tc dng ca enzyme ligase. Thnh phn phn ng ni:

4. To dng pICAmyQ/DH5a
c. Chn gen mc tiu vo vector biu hin pICAS1

4. To dng pICAmyQ/DH5a
Bin np v sng lc dng pICAmyQ/DH5a Vector ti t hp l kt qu ni ca vector pICAS1 v gen amyQ c bin np vo t bo ch DH5a vi tc nhn chn lc l kh nng khng ampicillin ca th bin np. Tch chit plasmid.

4. To dng pICAmyQ/DH5a
Kt qu bin np

5. To dng t bo pICAmyQ/MT 8-1

Chun b t bo nm men S. cerevisiae MT8-1 kh np Chng MT8-1 Nui cy lc qua m 300, sau cy dch trn vo mt YPD sao cho c OD610nm = 0,2. Lc 3h 300C 5ml mt YPD Ra sinh khi hai ln bng nc ct, huyn ph nh sinh khi trong 500ul dd TE/LiAc

Ly tm thu sinh khi 500v/pht, 10 pht, t0 phng

5. To dng t bo pICAmyQ/MT 8-1

Bin np vo t bo nm men S. cerevisiae MT8-1 100ul tb kh np, 1ul plasmid 70ul DMSO

Vortex k Lc nh 300C, 30

Gy sc nhit 420C, 15, yn trong nc 5

600ul PEG/TE/LiAc

Tri trn mt SD-trp, 3-4 ngy, 300C.

Ha trong 600ul TE 1X

Ly tm 10000v, 1 pht, thu sinh khi.

5. To dng t bo pICAmyQ/MT 8-1

Kim tra kt qu v s chn dng nm men mang vector ti t hp

Sau 3 4 ngy nui cy, chn cc khun lc nm men mc trn a mi trng.

Cy vo mi trng SD-tryp lng. Chn dng nm men c kh nng tng trng mnh trong mi trng SD-tryp.

5. To dng t bo pICAmyQ/MT 8-1 Kim tra kt qu v s chn dng nm men mang vector ti t hp

6. Phn tch dng nm men ti t hp pICAmyQ/MT8-1

Vector ti t hp pICAmyQ c bin np vo t bo nm men S.cerevisiae s st nhp vo NST ca nm men.

Kim tra bng cch tch chit DNA b gen ca nm men v dng lm khun mu cho phn ng khuch i gen amyQ vi cp mi c hiu.

Kt qu in di kim tra s hin din ca gen amyQ trong DNA b gen nm men ti t hp pICAS1/MT8-1

B. S biu hin ca gen amyQ trn b mt t bo nm men ti t hp

C. Kim tra s biu hin ca gen amyQ trn b mt t bo nm men ti t hp.

1. Nhum min dch hunh quang.

Kt qu nhum hunh quang min dch. A1: t bo pICAmyQ/MT8-1 nhum khng th c quan st trong nh sng thng. A2: t bo pICAmyQ/MT8-1 nhum khng th c quan st trong iu kin kch thch bc sng 495nm.

2. Kim tra hot tnh ca amylase.

pICAmyQ/MT8-1 Thu dch Lc 150v/p 300C, qua m

Ly tm 8000v/p

Thu cn

5ml mt YPD

2. Kim tra hot tnh ca amylase.

Tinh bt 0,5%

Sinh khi tb nm men

Sinh khi

Dch ni

C 3 ng nghim lc u, gi 500C, 5h

Sau , cho vo mi ng nghim 50ul dung dch lugol.

2. Kim tra hot tnh ca amylase.

Kt qu

Danh sch thnh vin

1. Nguyn Th Cm
2. Bi Th Khnh ng 3. L Th Quyn 4. Nguyn Xun V 5. inh Th Ngc Yn

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