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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

9/21/2010

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

9/21/2010

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

9/21/2010

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

9/21/2010

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

9/21/2010

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