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TOPIC
NI DUNG
TNG QUAN Saccharomyces cerevisiae (???!!!)
II
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
Vector YEp
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
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
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
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.
4. Enzyme ct gii hn
pICAS1
Thu nhn
pBluescript
bng pp SDS-kim Thu nhn pICAS1 bng pp SDS-kim
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
Thc hin bc 1 v 2 ba ln
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
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.
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
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
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
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
600ul PEG/TE/LiAc
Ha trong 600ul TE 1X
5. To dng t bo pICAmyQ/MT 8-1 Kim tra kt qu v s chn dng nm men mang vector ti t hp
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
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.
Ly tm 8000v/p
Thu cn
5ml mt YPD
Sinh khi
Dch ni
C 3 ng nghim lc u, gi 500C, 5h
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