Download - Biểu hiện gen nấm men
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CNG NGH GEN
GVHD: ThS. Nguyn Th Kim CcNHM : 3
LP: 52CNSH
TOPIC
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TNG QUANSaccharomyces cerevisiae (???! ! ! )I
II
TO DNG T BO NM MEN BIUHIN -AMYLASE CHU NHITTRN B MT T BO
NI DUNG
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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
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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.
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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
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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
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1
Vector st
nhp YIp
2
Vector YEp
2 loi vectorthngdng
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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
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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
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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
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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
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Cng ngh biu hin gen m haenzyme - amylase chu nhit
trn b mt t boSaccharomyces cerevisiae.
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A. Vt liu
1. Cc vi sinh vt
Escherichia coli DH5
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Saccharomyces ceereviseae MT8-1
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2. cc plasmid
Plasmid pICAS1
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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.
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Pbluescript
C kch thc 2958bp, chagen chn lc Amp
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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
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Cp mi khuch i gen amyQ
4. Enzyme ct gii hn
XhoI
NhoI
HincII
B Q trnh th c hin
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B. Quy trnh thc hin
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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
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1. To dng Ecoli pICAS1/DH5,pBluescript/DH5
Ch b E li kh
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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
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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
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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.
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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
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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.
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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/
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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
3. To dng pBAmyQ/DH5a
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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 T d BA Q/DH5
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3. To dng pBAmyQ/DH5a
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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
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e. Kt qu bin np
3. To dng pBAmyQ/DH5a
4 T d ICA Q/DH5
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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
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4. To dng pICAmyQ/DH5a
4 T d ICA Q/DH5
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4. To dng pICAmyQ/DH5a
Kt qu in di
4 T d ICA Q/DH5
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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. To dng pICAmyQ/DH5a
4 T d ICA Q/DH5
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c. Chn gen mctiu vo vector
biu hin pICAS1
4. To dng pICAmyQ/DH5a
4 To dng pICAmyQ/DH5a
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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
4 To dng pICAmyQ/DH5a
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Kt qu bin np
4. To dng pICAmyQ/DH5a
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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
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5. To dng t bo pICAmyQ/MT 8-1
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.
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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
5 To dng t bo pICAmyQ/MT 8-1
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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
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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
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Kt qu in di kim tra s hin din ca genamyQ trong DNA b gen nm men ti t hp
pICAS1/MT8-1
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B. S biu hin ca gen amyQ trn
b mt t bo nm men ti t hp
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C. Kim tra s biu hin ca
gen amyQ trn b mt t bonm men ti t hp.
1 Nhum min dch hunh quang
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1. Nhum min dch hunh quang.
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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
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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
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2. Kim tra hot tnh ca amylase.
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
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2. Kim tra hot tnh ca amylase.
Kt qu
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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