Biểu hiện gen nấm men

7/28/2019 Biu hin gen nm men

1/60

CNG NGH GEN

GVHD: ThS. Nguyn Th Kim CcNHM : 3

LP: 52CNSH

TOPIC


2/60

TNG QUANSaccharomyces cerevisiae (???! ! ! )I

II

TO DNG T BO NM MEN BIUHIN -AMYLASE CHU NHITTRN B MT T BO

NI DUNG


3/60

L sinh vt nhn chun, c th nui cy vi quym ln, cu trc gen c gii trnh vo nm1996 kch thc 1,35107

I. Tng quan1. cimnm men


4/60

C c tnh khng gy

bnh, khng tonict,c qu trnh ngdng ludi trong thcphm nn

S.cerevisiaecxp vonhm sinh vt an tonGRAS.

So vit bo hunh, tbo nm men sinh trngtngi nhanh.


5/60

C khnngto sinh khiln, thao tc ditruynngin, protein snphmctit

ra mi trng nn thu hid dng.

C cc cibin sau dch m (ng ha,phosphoryl ha) to protein c yhottnh sinh hc


6/60

2. Cc loi vector

Huht cc vectorsdng trong t bonm men l nhng vectorvnchuyn m nc th sao chp trong cE.coliv

S.cerevisiae.

Cc loi vector: Vector st nhp YIp Vector YEp (plasmid 2 m) Vector YRp Vector YCp

Vector YAC


7/60

1

Vector st

nhp YIp

2

Vector YEp

2 loi vectorthngdng


8/60

Vector st nhp YIp

Vector st nhp c tnh bnvng cao, chmt 1% qua 1 thh, khng cn p lcchnlc.

S st nhp vector vo b gen canmmen xy ra nhs trao i cho gia vectorv nhimscthto nn 2 dng DNA tngng mang gia n l trnh t DNA cavector.

Tns st nhpkhong 1-10 thbin


9/60


10/60

vector YEp (plasmid 2 m)

S. cerevisiae c thcha plasmid t nhin gil plasmid 2 m, c kch thc 6318bp, sbnsao trong t bo khong 50-100, chim 1-2%tng DNA

Plasmid 2 m csdngthitk ccvectort sao chp clp trong t bo nm menl YEp.

Tnsbinnpca plasmid rt cao, t 104105thbinnp/g DNA, thngrtbnvng,

chbothi mc 1% trong mtt bo


11/60


12/60

Cc gen m ha cho cc enzyme cn thit choqu trnh sinh tng hp cc cht dinh dngthit yu cho t bo.

Cc gen chn lc tri c tm thy t gen cc t bo ch nm men hoang di nh CUP1hoc t nm men bin i di truyn nh G418R .

3. Gen chnlc


13/60

Cc promoter c nhn bit bi RNA

polymerase ca t bo nm men chia lm 2 vngchc nng

Nhn t TATA

- ng vai tr trongvic xc nhv tr khiu phin m.

- S phin m thngthngbtutnucleotide th 30 sau

trnh t TATA.

Nhn tiu ha

thnglu

-Nm thnglunhn t TATA.

- L v tr gn ccprotein iu ha

4. Promoter


14/60


15/60

Cng ngh biu hin gen m haenzyme - amylase chu nhit

trn b mt t boSaccharomyces cerevisiae.


16/60

A. Vt liu

1. Cc vi sinh vt

Escherichia coli DH5


17/60

Saccharomyces ceereviseae MT8-1


18/60

2. cc plasmid

Plasmid pICAS1


19/60

Plasmid pKTH10

- C kch thc 6,8 kbp,mang gen khng

neomycine v on 2,2kbcha gen amyQ m hacho enzyme -amylase tB.amyloliquefaciens.

- Plasmid pKTH10 ny

c cung cp bi gios ngi c.


20/60

Pbluescript

C kch thc 2958bp, chagen chn lc Amp


21/60

Taq DNA polymerase.

Pfu Turbo DNA polymerase.

10X PCR buffer: dng cho Taq polymerase (KCl500mM, MgCl2 15mM, Tris-HCl 100mM, pH9).

10X Pfu PCR butffer.

dNTP.

3. Thnh phn pn ng PCR


22/60

Cp mi khuch i gen amyQ

4. Enzyme ct gii hn

XhoI

NhoI

HincII

B Q trnh th c hin


23/60

B. Quy trnh thc hin


24/60

Chun b E.coli kh np (DH5)

pICAS1

Thu nhn pKTH10

PCR vicp mic hiucho

amyQ

Cp miny tov tr ctXhoI v

NcoI

XhoI NcoI

amyQ

Thu nhnpICAS1

bng ppSDS-kim

Thu nhnpBluescriptbng ppSDS-kim

Chun b vector v DNA ch


25/60

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

Ch b E li kh


26/60

a. Chun b E.coli kh np

50ul E.coli DH5

5ml LB lng

Lcqua

m370C

500ul dch nui cy

5ml LB lng

Lctrong

2-3h

370C

Ly tm thu sinh khi

13000v trong 20 pht, 40C

Ra sinhkhi thucbng 1mlCaCl2,

100mM,

lnh

13000

vng

trong

20

pht,

40C

Ly

tm

thu

sinh

khi

Huyn ph sinh khitrong 1ml CaCl2, trong t nht 1h

Thc hin bc 1 v 2 ba ln

1

2

Bo qun bng glycerol80%, - 800C

b Bin np plasmid vo E coli DH5


27/60

b. Bin np plasmid vo E.coli DH5

Mt LB 2% agar + Apicillin: 50ug/ml

vo a petri

1ul plasmid

50ul dd CM 1X

50ul tb E.coli DH5a kh np

t trong nc 5

Gy sc nhit 42C 42

lnh nc 5

Tri trn a mt LB c Ampicillin

37C trong 16h

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


28/60

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 spht 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 LBlng 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.


29/60

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


30/60

2. Chun b plasmid mang gen chPlasmid pKTH10 mang gen amyQ thu c lm khun mu, thc hin phn ngPCR 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.


31/60

3. To dng pBAmyQ/DH5a

a. Chun b DNA gen mc tiu.

- Gen amyQ c khuch i t khunmu l pKTH10 bng phn ng PCR.

- Cp mi c hiu cho gen c thitk to v tr ct cho enzyme ct gii hnl XhoI v NcoI.

on gen c mang cc trnh t ctchuyn bit cho enzyme ct gii hnXhoI v NcoI.

Q/


32/60

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 muct.

b. Chun b DNA vector dng ha pBluescript

Ct bng HincII



33/60

c. Chn gen amyQ c vo vector pBluescript bng

phn ng ni di tc dng ca ligase.

Thnh phn phn ng ni:


3 T d BA Q/DH5


34/60



35/60

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.



36/60

e. Kt qu bin np


4 T d ICA Q/DH5


37/60

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 plasmidpICAS1 m vng v gen amyQ c ct gii hn hai u.

Dng b kit SephaglasTMBand Prep tinh sch mu ct.

4 T d ICA Q/DH5


38/60


4 T d ICA Q/DH5


39/60


Kt qu in di

4 T d ICA Q/DH5


40/60

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 enzymeligase.

Thnh phn phn ng ni:


4 T d ICA Q/DH5


41/60

c. Chn gen mctiu vo vector

biu hin pICAS1


4 To dng pICAmyQ/DH5a


42/60

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 thbin np.

Tch chit plasmid.


4 To dng pICAmyQ/DH5a


43/60

Kt qu bin np



44/60

Chun b t bo nm men S. cerevisiae MT8-1 kh np

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

5ml mt YPD

Chng MT8-1

Nui cy lc quam 300, sau

cy dch trn vomt YPD sao choc OD610nm = 0,2.

Lc 3h 300C

Ly tm thu sinh khi

500v/pht, 10 pht, t0 phng

Ra sinh khi hai

ln bng nc ct,huyn ph nhsinh khi trong500ul dd TE/LiAc


45/60


Bin np vo t bo nm men S. cerevisiae MT8-1

600ul PEG/TE/LiAc

100ul tb kh np, 1ul plasmid

Vortex k

Lc nh 300C, 30

70ul DMSO

Gy sc nhit

420C, 15, yn trong

nc 5

Ly tm

10000v,

1 pht,

thu sinh

khi.

Ha trong

600ul TE 1X

Tri trn mtSD-trp, 3-4ngy, 300C.


46/60

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

Sau 34 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


47/60

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

5. To dng t bo pICAmyQ/MT 8 1

6 Ph t h d ti t h


48/60

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

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

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

Kt qu in di kim tra s hin din ca gen


49/60

Kt qu in di kim tra s hin din ca genamyQ trong DNA b gen nm men ti t hp

pICAS1/MT8-1


50/60

B. S biu hin ca gen amyQ trn

b mt t bo nm men ti t hp


51/60


52/60


53/60


54/60

C. Kim tra s biu hin ca

gen amyQ trn b mt t bonm men ti t hp.

1 Nhum min dch hunh quang


55/60

1. Nhum min dch hunh quang.


56/60

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 Ki t h t t h l


57/60

2. Kim tra hot tnh ca amylase.

5ml mt YPD

pICAmyQ/MT8-1

Lc

150v/p300C,

qua

m

Ly tm 8000v/p

Thu dch

Thu cn

2 Kim tra hot tnh ca am lase


58/60


Sinh khi

Tinh bt 0,5%

Dch niSinh khi tb nm men

C 3 ng nghim lcu, gi 500C, 5h

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

2 Ki t h t t h l


59/60


Kt qu


60/60

1. Nguyn Th Cm 52130417

2. Bi Th Khnh ng 52130461

3. L Th Quyn 52130438

4. Nguyn Xun V 52130456

5. inh Th Ngc Yn 51230459

Danh sch thnh vin

Biểu hiện gen nấm men

Documents

Transcript of Biểu hiện gen nấm men