Ch04基因工程的常用载体-质粒载体

parA parA parA
parAparB parBparA
2. par
1. 1 1 ori 2 3
Plasmid cloning vector ori
2
GAATTC CTTAAG
ori
Ampr, Kanr, Tetr, Strr, Neor, Cmlr
2. 1 pSC101 9263 bp Col E1 6646 bp 2 pSC101 EcoRI, XhoI, PvuII, SacII, HpaIMlu I 3 pSC101 Tetr ColE1colicin E1
885 442
287
pUCMCS lacZ pUC19
5 AAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCGGGGTACCGAGCTCGAATTC3
HindIII SphI PstI SalI AccI HincII XbaI BamHI SmaI XmaI KpnI SacI EcoRI
9256
8597 7916
XhoI(8188) PvuII(7794) SacII(7565) MluI(7182)
EcoRI(9262) HindIII(30) 86 BamHI(376) SalI(652) 1276 pSC101 9263 bp 4051 2936 HpaI
7242 6223 repA
O
S
N H
O OH3C CH3 O OP O P O N H OOOH
N O N N
N
OH
O + N OHN
OH
Cl Cl O
Acetyl-CoA
O OH O P OO-
3kanamycinKan
NH3+ HO HO O HO HO O +H N 3 O O OH NH3+ OH OH NH3+
16 S rRNAmRNA
4pBR322
mobrop
5pBR322 pBR325: HaeII S1 4566 EcoRI
EcoRI(4779) EcoRI HindIII HindIII 86 BamHI SalIBamHI
5225
phage P1 Cmr DNA 4155ScaI (A/G) GCGC(T/C) (T/C)CGCG (A/G)
DNA
colici
n
imm
lysis
Colicin Colicin Colicin colicin
colicin
3. 1 Col E1
2antibiotics Ampr Kanr Tetr Strr Cmlr
2 1AmpicillinAmp
NH2 O H N O H N O S OH
DD
PstI ScaI PstI
pBR322 pBR325 4361 bp 5996
SalI
1276 3140 1917 2521 2108
pAT153: mobrop pBR322703 bprop 1.5-3 86 3450
Sca II Sca Pst II Pst Eco EcoRI RI HindIII dIII Hin BamHI HI Bam Sal Sal II
5273
62
EcoRI(6645) ScaI(6440) SmaI(5354) ScaII(5151) BtgI(5148)
601
1190 Col E1 6646 bp
NgoMIV(2142) NaeI(2144) ClaI(2311)
5140
BclI(4646) SnaBI(4255) MluI(3898)
1gcgcccaata 51gcagctggca 101gcaattaatg 151tatgcttccg 201acacaGGAAA 251gtcgactcta 301TTTTACaacg 351cttgcagcac 401caccgatcgc 451tgatgcggta 501tggtgcactc cgcaaaccgc cgacaggttt tgagttagct gctcgtatgt CAGCTATGAC gaggatcccc tcgtgactgg atcccccttt ccttcccaac ttttctcctt tcagtacaat ctctccccgc cccgactgga cactcattag tgtgtggaat CATGattacg gggtaccgag gaaaaccctg cgccagctgg agttgcgcag acgcatctgt ctgctctgat
2.pSC101 repA repArepA repA ori(250 bp)
par par A-T RNA Pol repA
repA
R1 R2 R3 R4 R5 R5
DnaA
mRNA(1kb)
repA
1. Partitioning parparAparB RNA Pol promoter par operon parB parB parB
M13 M13
attcattaat tgagcgcaac ctttacactt taacaatttc atgcctgcag CTGGCCGTCG acttaatcgc aagaggcccg gaatggcgcc acaccgcata taagccagcc
gcgttggccg aagcgggcag gcaccccagg tgtgagcgga ccaagcttgc ctcgaattca gcgttaccca cgtaatagcg cctgaatggc gcggtatttc gccgcatagt
H+
H+
Mg2+
Tet R
Tet O
Tet A
3
(+)
(-)
4. 1pBR322 1977Boyer F. BolivarR.L. Rodriguez 1 ori pMB1Col E1 Ampr RSF2124Ampr Tetr pSC101Tetr
EcoRI
(Tn3)Ampr, Cmlr, Strr Sur, Kanr
ScaI(3844) PstI(3607)
3293
pBR322 4361 bp
1276 3138 2519
NdeI(2295)
2106
1915
3pBR322 PstI
ScaI PstI EcoRI HindIIIClaI BamHI SalI
Βιβλιοθήκη Baidu
pBR322 4361 bp
+Tet
+Amp
4361 bp >10 kbDNA 1000~3000 copy mobmobilization
1. Vectors DNADNA 2. 1 2 3 4 5
3. 5 plasmid DNAM13 phage DNA phagemid cosmid
YAC BAC virus
Plasmid
DNA
1.
1200 kb
2. 2-3: 3. DNA
4. 1DNAcccDNA)
4StreptomycinStr
O CH3 HO HO HO H HO O O HN CH 3 O HO O N H2N OH N OH NH2 NH2 NH2
16S rRNAfmet-tRNA
Strr 30S
NH2 C C N HC O5' C C --P-O --P-O -CH 2 O O N O -P-O O N O- O- O H C 1' 4'C H H C C H OO= = = -
2590
pBR322 pAT153 4361 bp 3658
2435 1816
1276
2pUC University of CaliforniaJ. MessingJ. Vieria pBR322M13mp71982 1pUC lacZ lacZ- blue/white screening MCS polylinker Tetr
Kanr
NH2 C
ATP
O--P-O O--P-O-CH2 O--P-O - OO O OOO5'
N HC O H C N
C C
N C
= -
= -
NH3 HO O
O HO HO O +H N 3 O
= -
N
4'C H
H C
+
C 1' H
OH H
3' 2'
OH OH NH3+
O OH NH3+
2DNA open circular ocDNA 3DNA linear lDNA
scDNAl DNAocDNA
OC L SC
1. 1 Conjugative plasmids tra
2 Non-conjugative plasmids
2. 1F 2R 3Col bacteriocins 4 Degradative plasmids 5 Virulence plasmids Ti tra
pMB8 pMB1 pMB3
1/4361
R1 drd19
pBR313 pSC101 pMB9 pBR312
pBR322 pMB1 3145
1763
Ampr
2pBR322
RSF2124 Col E1
40 4153
86
EcoRI(4359) HindIII(29)ClaI(23) BamHI(375) SalI(651)
pUC19lacZMCS
atgaccatgattacgccaagcttgcatgcctgcaggtcgactctagaggat M T M I T P S L H A C R S T L E D ccccgggtaccgagctcgaattcactggccgtcgttttacaacgtcgtgac P R V P S S N S L A V V L Q R R D tgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccct W E N P G V T Q L N R L A A H P P ttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaa F A S W R N S E E A R T D R P S Q cagttgcgcagcctgaatggcgaatggcgcctgatgcggtattttctcctt Q L R S L N G E W R L M R Y F L L acgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatc T H L C G I S H R I W C T L S T I tgctctgatgccgcatagttaagccagccccgacacccgccaacacccgct C S D A A
1900 2519
2pUC19
1745
pUC19 2686 bp
HindIII(233) SphI(243) PstI(249) SalI(251) AccI(251) HincII(251) XbaI(257) BamHI(263) SmaI (268) XmaI(268) KpnI(272) SacI(278) EcoRI(284)
3. 1stringent plasmid 13 2relaxed plasmid 1060
1. RNACol E1 RNAII RNAIRNAII RNAI(108 nt) 5 3
RNAI RNAI RNAII RNAII Pol I
origin 3 5
RNaseH
RNAII(555 nt)
ATP
N
O CH3
HO HO HO
OH H
3' 2'
H HO O O HN CH 3
O HO O N H2N
OH N OH NH2 NH2 NH2
5TetracyclineTet
CH3 HC HO CH3 3 N OH OH OH O NH2 O
OH O
30StRNAA
pBR322
H+ 2+ Mg Mg2+
DD PBP penicillin-binding proteins
-beta-lactamase Ampr- -
NH2 H N O
H S HN OH O
OOH CO2
2ChloramphenicolCml
OH OH Cl O+ N OHN O Cl
50S23s rRNA
Cmlr
NH2 O
lacZ
pUC18
5GAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTT 3
EcoRI SacI KpnI BamHI XbaI SalI AccI HincII PstI SphI HindIII
SmaI XmaI
3pUC19lacZ_MCS lacZ_ lacZ_ promoter MCS(multiple cloning sites)
2132
3733
colicin E1 operon SOS
SOS Imm
Pc LexA
colicin
C C C
pi
imm
lysis
RecA RNA Pol DNA damage
C
colicin EcoRI colicin colici n
imm
lysis lysis imm colicin
Colicin Colicin Colicin colicin