1 9/21/2010 techniques in molecular biology 1. 2. 3. 4. 5. 6. 7. 8. electrophoresis: usual gel ~~...
TRANSCRIPT
1
9/21/2010
Techniques in Molecular Biology
1.
2.3.4.
4.5.6.7.8.
Electrophoresis:
Usual Gel ~~ rutinely usedPFGE ~~ Physical mappingDGGE~~SDS-PAGE
Restriction EnzymesHybridization: DNA, RNA, Protein, FISHCloning Strategy:
Genomic libraryMetagenomic library
DNA SequencingSynthesis of oligonucleotide, Directed mutagenesisPolymerase chain reactionDetection of gene expression (Northern, Western, Microarray, RNA tailing)Gene arrangement analysisPrimer ExtensionDNAse I Foot PrintingSequential Deletion analysis
Molecular cloning
Molecular cloning can be divided into several steps:
1.
2.
3.
Isolation and fragmentation of the source DNA.This can be total genomic DNA,DNA synthesized from RNA by reverse transcriptase,DNA synthesized by PCR.Digestion with restriction enzyme~~genomeJoining the DNA fragments to a cloning vector.
LigaseSticky endBlunt end
Introduction and maintainance of recombinant DNAin a host strain.
Method: Transformation
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2
Molecular cloning
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Insertional Inactivation(pBR322)
1.2.3.4.5.
It is relatively small, 4361 bpIt is stably maintained in E. coli (20-30 copies)It is easy to isolate in the supercoiled form<<<10 kb foreign DNA can be insertedThe complete squence of this plasmid hasbeen known
6. The unique site: PstI, SalI, EcoRI, HindIII,BamHI.
7. It has a gene conferring ApR and TcR
3
4
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pBR322(4361 bp)
pBR322 represents an earlygeneration of cloning vectorconstructed in vitro.
“Insertional inactivation”
TcR TcS
5
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Blue-White Selection(pUC19 or 18)
Cells carrying nomodified pUC19/18 are grown in the presentIPTG, Lac I can not bind promoter-operator lacZ’. LacZ’ pro-tein combaines with protein that is encoded by chromoso-mal DNA to form active hybrid B-galactocidase.
X-Gal (substrate)Blue product(blue colonies)
Hybrid B-Galactosidase
Cells that carry a plasmid-cloned DNA construct producewhite colonies on the same medium.
X-Gal (substrate)No Blue product(white colonies)
Hybrid B-Galactosidase
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ATGCAGAAATCATTGCTCATTCAACCGGCCAAATGCACCGGCTGCCGCCAGTGCGAGATGGCATGCTCGTTCGAGAAGGAGCGCAGCTTCAACCCGTCGAAGTCGCGCATCCGCGTCTTCGACATTCATTCCGAAGCCCG140
M Q K S L L I Q P A K C T G C R Q C E M A C S F E K E R S F N P S K S R I R V F D I H S E A R
CTTCGTCCCCTATACCTGCACCCAGTGCGCCCAGGCCTGGTGCCTGCAGGCCTGCCCGGTGGACGCCATCGGGATCGATGCCGTTACCAGGGCCAAGGTGGTCAACGACAACATCTGCGTCGGCTGCAAGGTCTGCACCA280
F V P Y T C T Q C A Q A W C L Q A C P V D A I G I D A V T R A K V V N D N I C V G C K V C T
TCGCCTGCCCCTTCGGCACCATCAACTATGTGGCCGACAGCGGTAAGGTGGCCAAGTGCGACCTGTGCGGCGGCGATCCCGCCTGCGCCAAGGCCTGTCCCACCGGGGCGATCACCTACGTGGATGCCGAGCAGACCGGC420
I A C P F G T I N Y V A D S G K V A K C D L C G G D P A C A K A C P T G A I T Y V D A E Q T G
560
Y D K M R A W A M K T D T Q S H T H A * M S W T G K F L R I D L T N G S V K T E E
TGAACCGCGCCTGGGCGCGGCAGTATCTGGGGCAGCGCGGCCTGGCCACCAAGTATTTCGCCGAGGAGGTCGACCCCAAGGTCGATCCCCTGTCGCCCGCCAACAAGATGATCTTCACCACCGGGCCGCTGACCGGAACC700
L N R A W A R Q Y L G Q R G L A T K Y F A E E V D P K V D P L S P A N K M I F T T G P L T G T
GCCGCCTCTACCGGCGGGCGCTATTCCGTGGTGACCAAGGGGCCGCTGACCAACTGCATCGCCTGCTCCAATTCCGGCGGCTTCTTCGGCAATGAGCTGAAGAACGCCGGCTGGGACATGATCATCGTGGAAGGCAGGTC840
A A S T G G R Y S V V T K G P L T N C I A C S N S G G F F G N E L K N A G W D M I I V E G R S
GCCCAAGCCCGTCTACCTTTCCATCGAGAACGAGACGGTGGAAATCCGCGACGCCGCCGAATTCTGGGGCAAGACGGTGTGGGAGACGGAGAACGGCCTGAAGGCCCGGCATCAGGACCCCATGCTGCGCGTCGCCACCA980
P K P V Y L S I E N E T V E I R D A A E F W G K T V W E T E N G L K A R H Q D P M L R V A T
TCGGCGCCGCCGGCGAGAAGGGCGTGCTGTATGCCTGCATCGTCAACGACCTGCACCGCGCCGCCGGGCGTTCGGGCGTGGGCGCGGTGATGGGGTCCAAGAACCTCAAGGCCATCGCGGTGCGCGGCACCAGGGGCGTG 1120
I G A A G E K G V L Y A C I V N D L H R A A G R S G V G A V M G S K N L K A I A V R G T R G V
ACGGTCAAGGACCCCGACCGCTTCATCAAGGCCACCATCGAGCAGAAGAAGGTGCTGGCCGACAACGCCGTCACCGGCCAGGGCCTGCCCAAATACGGCACCCAGGTGCTGATGAACGTCATCAACGAGATCGGCGCCAT 1260
T V K D P D R F I K A T I E Q K K V L A D N A V T G Q G L P K Y G T Q V L M N V I N E I G A M
GCCGACGCGGAACTTCAAGGAAGTGCAGTTCGAGGGCGCCCATAAGATCTCGGCCGAGGCCATGCACGAGCCGCGCGCCACCGATGGCAAGGCCAACCTGGCCACCAATGGCGCCTGTTTCGGCTGCACCATCGCCTGCG 1400
P T R N F K E V Q F E G A H K I S A E A M H E P R A T D G K A N L A T N G A C F G C T I A C
GCCGTATCTCGCGCATGGACCCCGGCCACTTCTCCATCACCTCCCGGCCCCAGTACAAGGAGCCCTCGGGCGGCGTGGAATACGAGGCCGCCTGGGCCATGGGATCGGATTGCGGCGTCGACGACCTGGAGGCCTGCACC 1540
G R I S R M D P G H F S I T S R P Q Y K E P S G G V E Y E A A W A M G S D C G V D D L E A C T
TTCGCCAACTTCATGTGCAACGAGCACGGCATCGACCCCATCTCCTTCGGCTCGACCCTGGCGGCGGCCATGGAAATGTTTGAGATGGGCGTCATCACCAAGGAGCAGACCGGCGGCGTCGAACTGAAATTCGGCTCGGC 1680
F A N F M C N E H G I D P I S F G S T L A A A M E M F E M G V I T K E Q T G G V E L K F G S A
CGAGGCCCTGGTGAAGATGGCGGAACTGACCGGCAAGGGCGAGGGCTTCGGCCTGGAACTGGGCCAGGGCTCGCGCCGGCTGTGCGCCAAATACGGCCACCCCGAACTGTCCATGACGGTCAAGAGCCAGGAATTCCCCG 1820
E A L V K M A E L T G K G E G F G L E L G Q G S R R L C A K Y G H P E L S M T V K S Q E F P
CCTATGATCCGCGCGGCATCCAGGGCATGGGCCTGACCTACGCCACCTCCAACCGTGGTGCCTGTCACCTGCGCTCCTACACCGTGGCGTCGGAAGTGCTGGGCATCCCGTTCAAGAGCGATCCCCTGGCCACCGATGGC 1960
A Y D P R G I Q G M G L T Y A T S N R G A C H L R S Y T V A S E V L G I P F K S D P L A T D G
AAGGCCGCCCTGGTCAAGGCGTTTCAGGACGCCACGGCGGCGTTCGACGCCTCGGGCATCTGCATCTTCACCACCTTCGCCTGGAGCCTGGAGAATCTGGCGCCCCAGATCGACGCCGCCTGCGAGGGCGAGTGGACCCC 2100
K A A L V K A F Q D A T A A F D A S G I C I F T T F A W S L E N L A P Q I D A A C E G E W T P
CGAAATCCTTCTCGAGGTGGGCGAGCGCATCTGGACCCTGGAACGCCAGTTCAATCTGGCGGCCGGCATGACGGCGGCGGACGACACCTTGCCCAAGCGCCTGCTGAAGGACGCGGCCAAGACCGGCCCGGCCAAGGGGC 2240
E I L L E V G E R I W T L E R Q F N L A A G M T A A D D T L P K R L L K D A A K T G P A K G
TGACCTCGGGCCTAGAAAAGATGCTGCCGGAATATTACCAGTTGCGCGGCTGGACCACGGACGGCGTGCCCACCACCGAGACCCTGAAGCGCCTGCAACTGGCCTGA2347
L T S G L E K M L P E Y Y Q L R G W T T D G V P T T E T L K R L Q L A *
RBS
3'-TTCGCGGACGTTG ACCGG GTG GTG GTG GTG GTG GTG ACT CCT AGG TGA TCA GGG-5'
6 X Histag * BamHI SpeI
Primer A
Primer B
5'-GGG ACT AGT TCT AGA ATGAGAAA TCATTGCTC-3'
SpeI XbaI (ORF1)
(ORF2)
TACGACAAGATGCGCGCCTGGGCGATGAAGACCGACACCCAGTCGCACACCCACGCTTGATTGAGAGGGCCTGGTCATGAGCTGGACTGGCAAGTTTCTCCGTATCGATCTCACCAATGGCAGCGTCAAGACCGAGGAAC
CGCTCCGGCCATCCTGGGCGCCATCCGCCACGCCACCGGCGTCACCATCCGCCAGGTCCCCGCCACCCCGGACCGGGTCCGCGCCGCCATCCGTGCCGCCAAGGGAGTTC 110
CGTCATGAGCGACGTCGTCGAAGCCGGCATCATCAATTGCGACGCCTGCCCGGTGCTGTGCCGCATCCGCGAGGGGCGTTCGGGGGCCTGCGACCGCTATGCCAATACCG 220
M S D V V E A G I I N C D A C P V L C R I R E G R S G A C D R Y A N T
GCGGCACGCTCACCCGCGTCGACCCGCTGGTGGTGGTCCAGGCCATCAAGGAGAAGGACGGCAAGCTGATCCCCTTCCTGTCCACCGACCGGGAATGGGACGGCGCCGTG 330
G G T L T R V D P L V V V Q A I K E K D G K L I P F L S T D R E W D G A V
GTCTCCAACTCGCCCACCTTCATCACCGGCATCGGCGCGTCGACCACTTATCCCGATTACAAGCCCGCCCCCTTCATCGTCGCGGCCGAGCATGACGGCGTCGACATGGT 440
V S N S P T F I T G I G A S T T Y P D Y K P A P F I V A A E H D G V D M V
CACCGTGGTGACCGAAGGCATCTTCAGCTATTGCGGCGTCAAGGTGAAGATCGACACCGACCGCTTCTTAGGCCCGGAACAGTCCACGGTGAGGGCGGGCGGCGAGGCGG 550
T V V T E G I F S Y C G V K V K I D T D R F L G P E Q S T V R A G G E A
TGGGCCATGTGACCACCAGCGAGTACGGCTCGCAAATGCTGTCCATCGGCGGCGTCCACCATCTGACCGGCGGCTCCAAGAAGGAGGGCAAGCTCACCTGCGACACCATG 660
V G H V T T S E Y G S Q M L S I G G V H H L T G G S K K E G K L T C D T M
CTGGCGCTGTGCAACAAGCAGGCGGTGGAGCTGACCATCGATAACGGCGCCACCGTGGTGGTCCAGGCGGGTCAGGCCCCCATCGTCAACGGCACCGTCGAGGAACGCAT 770
L A L C N K Q A V E L T I D N G A T V V V Q A G Q A P I V N G T V E E R M
GCGGGTGGGCTGCGGCTCGGCCACCATCGGCATGTTCGCCAAGCAATGGTTCGGGCTGGTGGAAGAGGTGGTGGTGGTCGACGACCACATCACCGGCCTGCTGTCCGAGC 880
R V G C G S A T I G M F A K Q W F G L V E E V V V V D D H I T G L L S E
ACAAGGCCGGACGCTATCTCGGCATCGAGGACAGCGGCGCGGTGATCCGCGGCCGCCGCTCGACGCTGGGGCGCTATTTCCAGGTGGCAGAGCCGGGGACCGGCTGGGGC 990
H K A G R Y L G I E D S G A V I R G R R S T L G R Y F Q V A E P G T G W G
GGCACCAACATCACCGACCCGCTGTCCATCCTCGACCCATTCAATCCCAAGGTGGCCAAGCCCGGCCATCGCCTGCTGATGGTCAGCACCACCGGCGAACATGCCGGCTA 1100
G T N I T D P L S I L D P F N P K V A K P G H R L L M V S T T G E H A G Y
TTACGTCCTGGATCAGGACCTGAAGCCGGTGGAGGCCGAGATGCCCGCCGCCGTGGCCGCCACGGTGGAGCGCATCCGCGAGAATTGCGAGGCGGCGCTGTGCTCGGTGG 1210Y V L D Q D L K P V E A E M P A A V A A T V E R I R E N C E A A L C S V
TGTTCATCGCCGGAGCGGGCGGCTCGCTGCGGGCCGGGGTCACCGAGAACCCGGTCAAGCTGACCCGCTCGGTCAAGGACTCACTGACCCTGGTCACCAGCGGCGGCGCC 1320V F I A G A G G S L R A G V T E N P V K L T R S V K D S L T L V T S G G A
CCGGTCTATGTCTGGCCGGGCGGCGGCATCACCTACATGGTCGACGTGCTGCGCATGCCCGACCACTCGTTCGGCGCCGTGCCCACCCCCGCTTTGGTGGCGCCCATCGA 1430P V Y V W P G G G I T Y M V D V L R M P D H S F G A V P T P A L V A P I E
GTTCACCATGCGCCTCGACGACTATCGCGCCCTGGGCGGCCACATGGATGCCGTGGTGCGGCTGGAGCAGGTCATCAATCAGGATCGGCGCAAATCGGTGCAATGGGACC 1540F T M R L D D Y R A L G G H M D A V V R L E Q V I N Q D R R K S V Q W D
CGGACAATCCCTGGCCCATGCTGAAGCGGAACTACAAGTGGGGGGACGACACGTGACCCCGCCCGTTGCCGCCCTGTTGCCCGACGGCAAGCGCCTGCACCTCCAGCACG 1650P D N P W P M L K R N Y K W G D D T
RBSStart scsA
. (Stop)
primer
6
Figure . DNA sequence and amino acid sequence deduced from ORF1
and ORF2 used for cloning in plasmid pUMP16
ORF38 Sequence
primer
▼ ▼
7
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ORF38
M 1 2 3 4 5
43
30
20
94
67
(kDa) RM(kDa)
A
(A) SDS=PAGE.
45
30
20.1
14.3
B
(B) Western Botting
M.
1.
2.
3.
4.
5.
RM.
Protein Marker (in kDa)
Total protein of uninduced cell
Total protein of induced cell
Soluble lysate of protein ORF38
Purified of protein ORF38
Western Blot of purified protein
Protein Rainbow Marker
ORF38 (~1.5 kb)
YtsA
4840
35
Response regulator
YvcQ
YxdJ YtsB
3730
29
Sensor kinase
YvcR
YxdK YtsC
5748
49
ATP binding protein
YvcS
YxdL YtsD YxdM
3124
22
Permease
YvcP
genome
0
347o
304o
265o B. subtilis
yxdL yxdM
Responseregulator
Sensorkinase
ATP binding Permeaseprotein
yxdK
Two-componentsystem
yvcR yvcSyvcQyvcP
ytsC ytsDytsBytsA
ABC transporter
yxdJ
Analysis of Gene(s) Function
Amino acid identity (%)
OD
600
ytsA Probe
M 1 2 3 4 5
4.7
2.6
1.8
1.0
ytsC Probe
M 1 2 3 4 5
0.1
0.01
1
10
1
23
4 5
1 3 11Incubation time (hr)
LB medium, 37oC
5 7 9
yxdJ Probe
M 1 2 3 4 5
yvcP Probe
M 1 2 3 4 5
yvcR Probe
M 1 2 3 4 5
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The ytsABCD, yvcPQRS and yxdJKLM constituteoperons
4.7
2.6
1.8
1.0
RNA transcript covering all genes on operon
RNA transcript covering two-component
system genesResponse
regulator
kb
4.7
2.6
1.8
1.0
kb
4.7
2.6
1.8
1.0
kb
4.7
2.6
1.8
1.0
yxdL Probe
M 1 2 3 4 5
kb4.7
2.6
1.8
1.0
kbkb
Sensor
kinase
ATP binding Permease
protein
kan
tet
pNO41
Response regulator over-expressing strains
Response regulator: ytsA, yvcP or yxdJ
bla
ori(pBR322)
ori(S. aureus)
transformationB. subtilis
8
terminatorPtet
terminator
M 1 2 3
kb
4.7
2.6
1.8
1.0
0.5
M 1 2 3
ytsC-probe
kb
4.7
2.61.8
1.0
0.5
M 1 2 3kb
4.7
2.61.8
1.0
0.5
kb
4.7
2.6
1.8
1.0
0.5
ytsB-probe yvcQ-probe yvcR-probe
M 1 2 3
M 1 2 3
yxdK-probe
1.8
1.00.5
kb
4.7
2.6
M 1 2 3
yxdL-probe
1.8
1.0
0.5
kb
4.72.6
M: RNA marker
1 : wild type
2 : cells containing pNO41
9/21/2010
Overexpression of response regulator induced thetranscription of ABC transporter in the same operon
Response
regulator
Sensor
kinase
ATP binding Permease
protein
cat lacZ amyE front
blaamyE back amyE front
lacZcat
B. subtilis genome
9
pDL2
bla
amyE back
3 : cells containing pNO41-ytsA, yvcP or yxdJ
ABC transporter promoter-lacZ fusion strains
promoter region
ABC transporter gene
10
-57
-68
-78
-88
-106
-149
-204
ytsB lacZ
amyEback amyE
front
BSGY01
BSGY02
BSGY03
BSGY04
BSGY05
BSGY06
BSGY07
BSGY08
b-galactosidase activity
pNO41-ytsA
25
27
22
22
14
21
21
20
(Unit)
ATG +52
100
82
94
127
4522
3876
5264
6195
ytsC
pNO41
9/21/2010
The cis-acting sequence required for induction of ytsCtransporter expression by YtsA response regulator
755037
25
kDM 1 2 3 4 5 6
GST-YvcP
5 6M 1 2 3 4
75
5037
25
kD
GST-YtsA
M 1 2 3 4 5 6
75
50
37
25
kD
GST-YxdJ
-247
Purification of GST-YtsA, GST-YvcP and GST-YxdJresponse regulator proteins
M: protein marker1 : crude extract2 : pellet3 : supernatant4 : column flowthrough5 : column wash6 : column eluate
DZÇÃÉsÉNÉ`ÉÉǾå©ÇÈǞǽDžÇÕÅA
ÅgQuickTimeýÅhã@î\ägí£Ç²ÅAÅgPlanar RGBÅhêLí£ÉvÉçÉOÉâÉÄÇ™ïKóvÇ Ç ÅB
A C G T T G C A
ATGGGAGGATGCTGACTTCCTTTTTATAATAAAGAAAAAGGAGGAGCAGAACAT
TACCCTCCTACGACTGAAGGAAAAATATTATTTCTTTTTCCTCCTCGTCTTGTACTTGC
A Met (+1)
yvcQ-109 -93 -79
-51 -32 -12 yvcR
GACCCCTCGTGAAATGTGACAGCATTGTAAGATTGGGGAGCGGAATTGCAAGAAAGTTCG
CTGGGGAGCACTTTACACTGTCGTAACATTCTAACCCCTCGCCTTAACGTTCTTTCAAGC
9/21/2010
DNase I footprinting analysis of GST-YvcP binding toyvcR promoter
Induction positive Induction negative
GST-YvcP GST-YvcP
How the Un/culturable
microbes can be exploited
for biotechnological
applications?
11
9/21/2010
Metagenome ProjectMost microbes in environment are unculturable
99% can not be cultured
Under laboratory condition
How to exploit them?
EnzymesAntibioticsEtc.
Construction of metagenomicLibrary
Only 1% can be cultured
Under laboratory condition
How to exploit them?
Construction of genomicLibrary
Vector: Fosmid
CosmidBACplasmid
Metagenome
Meta: come from statistical analysisMeta-analysis: The process of statistically
Combining separate analyses
Genome: genetic materials of organisms
“Culture-independent of genomic analysisof microbial communities”
12
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13
Fig. Map of Bacterial Artificial Chromosome (BAC)
Construction of Metagenomic Library
Environmentalsamples
forVectorLibrary
LamdaCosmid
DNA fragment whichcan be packaged
13-15 kb
30-40 kb
Bacterial Artificial Chromosome (BAC)Yeast Artificial Chromosome (YAC)
120-300 kb250-400 kb
9/21/2010
Genomic Library (=Gene Bank)
“The whole of cloning collection which representeach DNA fragment from bacterial genomes”
APLICATION MICROBIAL GENETIC INECOLOGY STUDY
14
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15
Fig. Step in biodiversity analysis of microbialcommunity using phylogenetic probes
63f: 5-CAG GCC TAA
CAC ATG CAA GTC-3
1387r: 5-GGG CGG WGT
GTA CAA GGC-3
27f:
1392r:
PRIMER
Denatured Gradient Gel Electrophoresis(DGGE)
PCR amplification ------single band containing ampli-fied DNA, BUT band can contain many highly relatedbut not identical genes/DNA sequences.
GOAL: to sequence amplified genes (as in phylogenetic anal)NEED: additional step------DGGE“Method that separate genes of the same size that differin base sequence”. This technique employs a gradient of DNAdenaturant (Mixture of Urea and formamide)
DNA fragment moving through the gel reach a region conta-ining denaturant, the strand begin melt at which point migra-tion stop.
Different in melting-------different in base sequenceThus, different band in DGGE------different gene
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16
a
b
PCR
DGGE
DGGE: Denature Gradient Gel Electrophoresis
Fluorescent in situ Hybridization(FISH)
Fluorescently labeled nucleic acid probe
Target: rRNA
(Fluorescein,Tetramethylrhodamine)
Widely used in : microbial ecologyClinical diagnostic
Universal probeSpecific probe
Detection and identificationof bacterial cell in environment