Cloning Simulation Project

1
Cloning Simulation Project
Group F
Cloning of DexA Towards the degradation of
Dental Plaque
2
Group members
Kadi Tiong Mohd Hafizi Mansor Lye Kheng Yong Mohd
Nor Azim Ab Patar Murni Mohd Hanafi Zaid Nor
Azreen Zulkafli Nur Hafiza Md. Yusop Nur Shila
Mohamed Nor
Group F
3
Cloning of DexA Towards degradation of Dental
Plaque
4
OBJECTIVES

• To understand the concept of molecular biology
  and biotechnology introduced in GTB 204/3
  Molecular Biology Technique.
• To obtain the experiences in cloning strategy
  through simulation
• To expose ourselves to the finding from the
  concerned website and analysis the information
  that we get and organize our work so that to get
  our final product.

5
INTRODUCTION

• Dental plaque,
• the bacterial film adherent to tooth surfaces,
• is composed of closely packed bacteria and
  non-cellular material.
• lead to Dental caries - demineralization of the
  tooth surface cause by bacteria
• also known as tooth decay and is commonly called
  cavities.
• If decay is allowed to spread, it may penetrate
  the root and enter the pulp chamber, causing an
  abscess

6
How to remove Dental Plaque?

• Enzymes produced by Lipomyces starkeyi, that
  hydrolyze the ?-1, 3, ? -1, 4 and ? -1, 6
  linkages in the glucans, have recently been
  tested.
• Although these enzymes have been effective,
  their practical application on humans is not
  possible because L. starkeyi is a potential
  pathogen.

7
Dental plaque formation
8
Why Dextranase?

• For alternative , commercial enzyme preparations
  called Dextranase were chosen
• In view of their potential use as dental plaque
  control agent and as adjuncts to brushing and
  flossing for the removal of plaque and control of
  gingivitis.
• To verify the possibility of reducing or
  preventing such glucan formation, enzyme
  dextranase produced by the genus Penicillium were
  chosen for prevention.

9
Why Dextranase ?

• In the concentration range 3.7510-21.510-1
  U/l, was capable of blocking in vitro synthesis
  of the glucans catalyzed by glucosyltransferases
  of Streptococcus sobrinus.
• With small amounts of the enzyme, a limited
  production of glucans was observed, but the
  synthesized polysaccharides showed no adhesive
  properties.

10
Why Dextranase ?

• Dextranase was also able to degrade partially
  (about 20) pre-synthesized glucans.
• The stability characterization of the enzymes
  Dextranase in commercial mouthwash without
  ethanol and at physiological temperature (37C)
  showed that it is possible to use Dextranase as a
  dental plaque control agent.
• Beside that, enzymes Dextranase are stable at
  pH 6.0 and up to 40C. This enzyme
  preferentially cleaves the a-1, 6 linkages of
  dextran.

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Hunt for gene

• First of all, our group has come across an
  article at http//www.sciencedirect.com that
  talks about the degradation of dental plaque
  glucans by a type of enzymes called Dextranase.
• Then, we try to search of the enzyme sequence
  through the GenBank website at http//www.ncbi.nlm
  .nih.gov .
• .

13
Cont

• We have used various keywords for the purpose of
  searching the compatible DNA sequence of our
  enzyme, such as dextranase and penicillium
  funiculosum (organism that produce the dextranase
  enzymes).

14
Get the gene sequence from Genbank

• After long time of searching through the gene
  bank, we finally successfully obtain the gene
  sequence that code for the enzyme dextranase.
• The gene sequence is from an organism called
  Penicillium funiculosum.
• The accession number for this gene sequence is
  AJ272066 and it consists of 1851 base pairs.

15
The nucleotide sequence of gene of interest gene
is

• 1 atggccacaa tgctaaagct acttgcgttg acccttgcaa
  ttagcgagtc cgccattgga
• 61 gcagtcatgc acccacctgg cgtttctcat cccggtaccc
  atacgggcac tacgaataat
• 121 acccattgcg gcgccgactt ctgtacctgg tggcatgatt
  caggggagat caacacgcag
• 181 acacctgtcc aaccagggaa cgtgcgccaa tctcacaagt
  attccgtgca agtgagtcta
• 241 gctggtacaa acaactttca tgactccttt gtatatgaat
  cgatcccccg gaacggaaat
• 301 ggtcgcatct atgctcccac cgatccatcc aacagcaaca
  cattagattc aagcgtggat
• 361 gatggaatct cgattgagcc tagtatcggc ctcaatatgg
  catggtccca attcgagtac
• 421 agccaggatg tcgatataaa gatcctggca actgatggct
  catcgttggg ctcaccaagt
• 481 gatgttgtta ttcgccccgt ctcaatctcc tatgcgattt
  ctcagtccaa cgatggcggg
• 541 attgtcatcc gggtcccagc cgatgcgaac ggccgcaaat
  tttcagtcga attcaaaaat
• 601 gacctgtaca ctttcctctc tgatggcaac gagtacgtca
  catcgggagg tagcgtcgtc
• 661 ggcgttgagc ctaccaacgc acttgtgatc ttcgcaagtc
  cgtttcttcc ttctggcatg

16
721 attcctcata tgaaacccca caacacgcag accatgacgc
caggtcctat caataacggc 781
gactggggcg ccaagtcaat tctttacttc ccaccaggtg
tatactggat gaaccaagat 841 caatcgggca
actcgggtaa attaggatct aatcatatac gtctaaactc
gaacacttac 901 tgggtctacc ttgcccccgg
tgcgtacgtg aagggtgcta tagagtattt caccaagcaa
961 aacttctatg caactggtca tggtgtccta tcaggtgaaa
actatgttta ccaagccaat 1021 gctggcgaca
actatgttgc agtcaagagc gattcgacca gcctccggat
gtggtggcac 1081 aataaccttg ggggtggtca
aacatggtac tgcgttggcc cgacgatcaa tgcgccacca
1141 ttcaacacta tggatttcaa tggaaattct ggcatctcaa
gtcaaattag cgactataag 1201 caggtgggag
ccttcttctt ccagacggat ggaccagaaa tctatcccaa
tagtgtcgtg 1261 cacgacgtct tctggcacgt
caatgatgat gcaatcaaaa tctactattc gggagcatct
1321 gtatcgcggg caacgatctg gaaatgtcac aatgacccaa
tcatccagat gggatggaca 1381 tctcgggata
tcagtggagt gacaatcgac acattaaatg ttattcacac
ccgctacatc 1441 aaatcggaga cggtggtgcc
ttcggctatc attggggcct ctccattcta tgcaagtggg
1501 atgagtcccg attcaagcaa gtccatatcc atgacggttt
caaacgttgt ttgcgagggt 1561 ctttgcccgt
ccctgttccg catcacaccc ctacagaact acaaaaattt
tgttgtcaaa 1621 aatgtggctt tcccagatgg
gctacagaca aatagtattg gcacaggaga aagcattatt
1681 ccagccgcat ctggtctaac gatgggacta aatatctcca
gctggactgt tggtggacaa 1741 aaagtgacaa
tggagaactt tcaagccaat agcctggggc agttcaatat
tgacggcagc 1801 tattgggggg agtggcagat
tagtcgaatt tccagctctc agagcgcgtg a 
The nucleotide sequence of gene of interest gene
is
17
Look for the Open Reading Frame (ORF)

• Open Reading Frame (ORF) is a DNA sequence that
  containing series of codons, which can be
  translatable into protein.
• ORF starts with a start codon (ATG) and ends with
  a stop codon (TGA, TAG or TAA).
• Within the start codon and stop codon,
  containing the actual sequence that code for a
  polypeptide/protein. For the gene that code for
  the enzyme dextranase, the ORF can be defined by
  accessing through the website
• http//www.ncbi.nlm.nih.gov/gorf/gorf.html.

18
Cont.

• Within this website, there is a tool which helps
  us to search for the ORF within the interest
  gene, the ORF finder.
• The ORF finder is an analysis tool which help to
  find all the ORF within the users selected gene
  sequence.
• The gene bank accession number for dexA gene
  AJ272066 is entered and the analysis is performed.

19
Purpose of Doing ORF Analysis

• To confirm that the dexA gene sequence can
  exactly be translatable into expected functional
  protein.
• To know the length of the frame needed in this
  cloning process.

20
Result from the ORF analysis
21
1 atggccacaatgctaaagctacttgcgttgacccttgcaatt
agc
M A T M L K L L A L T L A I
S
46 gagtccgccattggagcagtcatgcacccacctggcgtttct
cat
E S A I G A V M H P P G V S
H
91 cccggtacccatacgggcactacgaataatacccattgcggc
gcc
P G T H T G T T N N T H C G
A
136 gacttctgtacctggtggcatgattcaggggagatcaacacg
cag
D F C T W W H D S G E I N T
Q
181 acacctgtccaaccagggaacgtgcgccaatctcacaagtat
tcc
T P V Q P G N V R Q S H K Y
S
226 gtgcaagtgagtctagctggtacaaacaactttcatgactcc
ttt
V Q V S L A G T N N F H D S
F
271 gtatatgaatcgatcccccggaacggaaatggtcgcatctat
gct
V Y E S I P R N G N G R I Y
A
316 cccaccgatccatccaacagcaacacattagattcaagcgtg
gat
P T D P S N S N T L D S S V
D
361 gatggaatctcgattgagcctagtatcggcctcaatatggca
tgg
D G I S I E P S I G L N M A
W
406 tcccaattcgagtacagccaggatgtcgatataaagatcctg
gca
S Q F E Y S Q D V D I K I L
A
451 actgatggctcatcgttgggctcaccaagtgatgttgttatt
cgc
T D G S S L G S P S D V V I
R
496 cccgtctcaatctcctatgcgatttctcagtccaacgatggc
ggg
P V S I S Y A I S Q S N D G
G
541 attgtcatccgggtcccagccgatgcgaacggccgcaaattt
tca
I V I R V P A D A N G R K F
S
586 gtcgaattcaaaaatgacctgtacactttcctctctgatggc
aac V E F K N D L Y T F
L S D G N
22
631 gagtacgtcacatcgggaggtagcgtcgtcggcgttgagcct
acc
E Y V T S G G S V V G V E P
T
676 aacgcacttgtgatcttcgcaagtccgtttcttccttctggc
atg
N A L V I F A S P F L P S G
M
721 attcctcatatgaaaccccacaacacgcagaccatgacgcca
ggt
I P H M K P H N T Q T M T P
G
766 cctatcaataacggcgactggggcgccaagtcaattctttac
ttc
P I N N G D W G A K S I L Y
F
811 ccaccaggtgtatactggatgaaccaagatcaatcgggcaac
tcg
P P G V Y W M N Q D Q S G N
S
856 ggtaaattaggatctaatcatatacgtctaaactcgaacact
tac
G K L G S N H I R L N S N T
Y
901 tgggtctaccttgcccccggtgcgtacgtgaagggtgctata
gag
W V Y L A P G A Y V K G A I
E
946 tatttcaccaagcaaaacttctatgcaactggtcatggtgtc
cta
Y F T K Q N F Y A T G H G V
L
991 tcaggtgaaaactatgtttaccaagccaatgctggcgacaac
tat
S G E N Y V Y Q A N A G D N
Y
1036 gttgcagtcaagagcgattcgaccagcctccggatgtggtgg
cac
V A V K S D S T S L R M W W
H
1081 aataaccttgggggtggtcaaacatggtactgcgttggcccg
acg
N N L G G G Q T W Y C V G P
T
1126 atcaatgcgccaccattcaacactatggatttcaatggaaat
tct
I N A P P F N T M D F N G N
S
1171 ggcatctcaagtcaaattagcgactataagcaggtgggagcc
ttc
G I S S Q I S D Y K Q V G A
F
1216 ttcttccagacggatggaccagaaatctatcccaatagtgtc
gtg F F Q T D G P E I Y
P N S V V
23
1261 cacgacgtcttctggcacgtcaatgatgatgcaatcaaaatc
tac
H D V F W H V N D D A I K I
Y
1306 tattcgggagcatctgtatcgcgggcaacgatctggaaatgt
cac
Y S G A S V S R A T I W K C
H
1351 aatgacccaatcatccagatgggatggacatctcgggatatc
agt
N D P I I Q M G W T S R D I
S
1396 ggagtgacaatcgacacattaaatgttattcacacccgctac
atc
G V T I D T L N V I H T R Y
I
1441 aaatcggagacggtggtgccttcggctatcattggggcctct
cca
K S E T V V P S A I I G A S
P
1486 ttctatgcaagtgggatgagtcccgattcaagcaagtccata
tcc
F Y A S G M S P D S S K S I
S
1531 atgacggtttcaaacgttgtttgcgagggtctttgcccgtcc
ctg
M T V S N V V C E G L C P S
L
1576 ttccgcatcacacccctacagaactacaaaaattttgttgtc
aaa
F R I T P L Q N Y K N F V V
K
1621 aatgtggctttcccagatgggctacagacaaatagtattggc
aca
N V A F P D G L Q T N S I G
T
1666 ggagaaagcattattccagccgcatctggtctaacgatggga
cta
G E S I I P A A S G L T M G
L
1711 aatatctccagctggactgttggtggacaaaaagtgacaatg
gag
N I S S W T V G G Q K V T M
E
1756 aactttcaagccaatagcctggggcagttcaatattgacggc
agc
N F Q A N S L G Q F N I D G
S
1801 tattggggggagtggcagattagtcgaatttccagctctcag
agc
Y W G E W Q I S R I S S S Q
S
1846 gcgtga 1851
A
24

• The whole gene sequence (1 frame) is chosen for
  the expression of the dextranase enzyme.
• 1 frame starts from base pair 1 and ends at
  base pair 1850 which has the length of 1851
  basses.
• The reading frame that is used determines which
  amino acids that will be encoded by a gene.

25
Determination of Amino Acids Sequence Molecular
Weight

• We have done the translation of the interest gene
  sequence to amino acids sequence.
• By using the Bioedit software.

26
Results
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29
Analysis of the sequence

• Length 1851 bp
• 1 amino acids 3 base pair 110 Dalton
• For 1851 bp,
• there would be 1851 / 3 617 amino acid
• Molecular weight of the dextranase
• 1851 / 3 x 110 Dalton
• 617 x 110 Dalton 67870 Dalton
• approximately 67 kDa

30
Look for the restriction enzyme sites

• Restriction Enzyme (RE)
• - A type of protein that help to recognize
  specific short nucleotide sequence.
• - It cut the DNA in a specific site that
  recognized by the RE mentioned above.

31
Analysis of the restriction enzymes for the dexA
gene

• To determine the restriction enzyme that is able
  to to cut the gene of interest that has been
  used.
• Go to the website http//www.firstmarket.com/cut
  ter/cut2.html

32
Results
 
Penicillium funiculosom
1851 base pairs
Graphic map Table by enzyme name BalI

MluNI
CfrI
HindII
atggccacaatgctaaagctacttgcgttgaccctt
gcaattagcgagtccgccattggagcagtcatgcaccca base
pairstaccggtgttacgatttcgatgaacgcaactgggaacgttaatc
gctcaggcggtaacctcgtcagtacgtgggt 1 to 75 EaeI
HincII
MscI


AccB1I
AccB1I BbiII BbeI
Asp718I
Eco64I Hsp92I Bsp143II Acc65I
BsrDI Hin1I EheI
SexAIcctggcgtttctcatcccggtacccatacgggcactacgaataa
tacccattgcggcgccgacttctgtacctgg base
pairsggaccgcaaagagtagggccatgggtatgcccgtgatgcttatt
atgggtaacgccgcggctgaagacatggacc 76 to 150
Eco64I
KasI Msp17I HaeII BanI KpnI
BanI NarI BsaHI
BshNI
BshNI AcyI BstH2I



PshAI
tggcatgattcaggggagatcaacacgcagacacctgtccaaccagg
gaacgtgcgccaatctcacaagtattcc base
pairsaccgtactaagtcccctctagttgtgcgtctgtggacaggttgg
tcccttgcacgcggttagagtgttcataagg 151 to 225





BspXI Bsp106I

BspDI BscI
BspHI ClaI BseCI
gtgcaagtgagtctagctggtacaaacaactttcatgactcctttgtat
atgaatcgatcccccggaacggaaat base
pairscacgttcactcagatcgaccatgtttgttgaaagtactgaggaa
acatatacttagctagggggccttgccttta 226 to 300
RcaI
BanIII
Bsu15I

Bsa29I
33




ggtcgcatctatgctcccacc
gatccatccaacagcaacacattagattcaagcgtggatgatggaatctc
gatt base pairsccagcgtagatacgagggtggctaggtagg
ttgtcgttgtgtaatctaagttcgcacctactaccttagagctaa
301 to 375






MflI

BstX2I gagcctagtatcggcctcaatatggcatgg
tcccaattcgagtacagccaggatgtcgatataaagatcctggca
base pairsctcggatcatagccggagttataccgtaccagggttaag
ctcatgtcggtcctacagctatatttctaggaccgt 376
to 450
XhoII

BstYI


FriOI

BanII DraIII Esp3I
actgatggctcatcgttgggctcaccaagtgat
gttgttattcgccccgtctcaatctcctatgcgatttctcag
base pairstgactaccgagtagcaacccgagtggttcactaca
acaataagcggggcagagttagaggatacgctaaagagtc 451 to
525 Eco24I
BsmBI



BsaBI Psp5II EclXI BstMCI
MamI EcoO109I
BstZI BsaOI ApoI
BsrBRI DraII CfrI Eco52I
EcoRI tccaacgatggcgggattgtcatccgggtcccagccga
tgcgaacggccgcaaattttcagtcgaattcaaaaat base
pairsaggttgctaccgccctaacagtaggcccagggtcggctacgctt
gccggcgtttaaaagtcagcttaagttttta 526 to 600
Bsh1365I EagI
BsiEI ApoI AcsI
Bse8I PpuMI EaeI Bsh1285I

XmaIII AcsI

Bsp1407I
BsrGI

gacctgtacactttcctctctgatggcaacgagtacg
tcacatcgggaggtagcgtcgtcggcgttgagcctacc base
pairsctggacatgtgaaaggagagactaccgttgctcatgcagtgtag
ccctccatcgcagcagccgcaactcggatgg 601 to 675
SspBI




34




FauNDI aacgcacttgtgatcttcgca
agtccgtttcttccttctggcatgattcctcatatgaaaccccacaacac
gcag base pairsttgcgtgaacactagaagcgttcaggcaaagaag
gaagaccgtactaaggagtatactttggggtgttgtgcgtc 676 to
750
NdeI


BbiII
AhdI Psp5II BshNI AcyI BstH2I
AcyI AspEI PpuMI
BanI NarI EheI XcmI
Hin1I EclHKI KasI Msp17I HaeII
SexAI AccIaccatgacgccaggtcctatcaataacggcg
actggggcgccaagtcaattctttacttcccaccaggtgtatac base
pairstggtactgcggtccaggatagttattgccgctgaccccgcggtt
cagttaagaaatgaagggtggtccacatatg 751 to 825
Msp17I DraII Eco64I Hsp92I
DraIII Hsp92I EcoO109I
AccB1I BbiII BbeI Bst11
BsaHI Eam1105I Hin1I BsaHI
Bsp143II
BcoI
Ama87I MflI

AvaI BstX2I
tggatgaaccaagatcaatcgggcaactcgggtaaattaggatc
taatcatatacgtctaaactcgaacacttac base
pairsacctacttggttctagttagcccgttgagcccatttaatcctag
attagtatatgcagatttgagcttgtgaatg 826 to 900
BsoBI XhoII
07I
Eco88I BstYI


Pfl23II
SunI
AccI
BsiWI BstSFI
tgggtctaccttgcccccggtgcgtacgtgaagggtgctatagag
tatttcaccaagcaaaacttctatgcaact base
pairsacccagatggaacgggggccacgcatgcacttcccacgatatct
cataaagtggttcgttttgaagatacgttga 901 to 975
SplI SfcI
PspLI

BsaAI




XcmI
ggtcatggtgtcctatcaggtgaaaactatgtttaccaagccaatgctg
gcgacaactatgttgcagtcaagagc base
pairsccagtaccacaggatagtccacttttgatacaaatggttcggtt
acgaccgctgttgatacaacgtcagttctcg 976 to 1050
35
BsaWI EcoT14I
BseAI
Kpn2I BssT1I
MroI AccIII Eco130I
gattcgaccagcctccgg
atgtggtggcacaataaccttgggggtggtcaaacatggtactgcgttgg
cccgacg base pairsctaagctggtcggaggcctacaccaccgtgt
tattggaacccccaccagtttgtaccatgacgcaaccgggctgc 1051
to 1125 Bsp13I StyI

BsiMI ErhI
BspEI




AcsI
atcaatgcgccaccattcaacactatggattt
caatggaaattctggcatctcaagtcaaattagcgactataag base
pairstagttacgcggtggtaagttgtgatacctaaagttacctttaag
accgtagagttcagtttaatcgctgatattc 1126 to 1200
ApoI




Bbv12I BbiII

Alw44I Msp17I Bbv16II BspMI
BstXI VneI AspHI
AcyI BpuAIcaggtgggagccttcttcttccagacggatggaccagaa
atctatcccaatagtgtcgtgcacgacgtcttctgg base
pairsgtccaccctcggaagaagaaggtctgcctacctggtctttagat
agggttatcacagcacgtgctgcagaagacc 1201 to 1275

ApaLI Hin1I AatII
Alw21I Hsp92I

BsiHKAI BsaHI BbsI


MslI
BcgI
cacgtcaatgatgatgcaatcaaaatctactattcgggagcatctg
tatcgcgggcaacgatctggaaatgtcac base
pairsgtgcagttactactacgttagttttagatgataagccctcgtag
acatagcgcccgttgctagacctttacagtg 1276 to 1350

BpiI



BsaBI Eco88I
MamI AvaI
MslI
BsrBRI BcoI EcoRV
aatgacccaatcatccagatgggatggacatctcgggatatcagtgg
agtgacaatcgacacattaaatgttatt base
pairsttactgggttagtaggtctaccctacctgtagagccctatagtc
acctcactgttagctgtgtaatttacaataa 1351 to 1425
Bsh1365I Eco32I

Bse8I Ama87I
BsoBI

36
AccB1I

Eco64I
Esp3I
DraII cacacccgctacatcaaatcg
gagacggtggtgccttcggctatcattggggcctctccattctatgcaag
tggg base pairsgtgtgggcgatgtagtttagcctctgccaccacg
gaagccgatagtaaccccggagaggtaagatacgttcaccc 1426 to
1500 BsmBI
EcoO109I
BanI
BshNI




MslI Psp1406I
atgagtcccgattcaagcaagtccatatccatgacgg
tttcaaacgttgtttgcgagggtctttgcccgtccctg base
pairstactcagggctaagttcgttcaggtataggtactgccaaagttt
gcaacaaacgctcccagaaacgggcagggac 1501 to 1575







BstSFI
AcsI
BstSFI ttccgcatcacacccctacagaactacaaaaattttgttgt
caaaaatgtggctttcccagatgggctacagaca base
pairsaaggcgtagtgtggggatgtcttgatgtttttaaaacaacagtt
tttacaccgaaagggtctacccgatgtctgt 1576 to 1650
SfcI ApoI
SfcI




MspA1I
GsuI

NspBIIaatagtattggcacaggagaaagcattattccagccgca
tctggtctaacgatgggactaaatatctccagctgg base
pairsttatcataaccgtgtcctctttcgtaataaggtcggcgtagacc
agattgctaccctgatttatagaggtcgacc 1651 to 1725

PvuII
BpmI





BstXI SspI
actgttggtggacaaaaagtgacaatggagaactttcaagccaata
gcctggggcagttcaatattgacggcagc base
pairstgacaaccacctgtttttcactgttacctcttgaaagttcggtt
atcggaccccgtcaagttataactgccgtcg 1726 to 1800
37
The following endonucleases were selected but
don't cut this sequence

• AatI, Acc113I, Acc16I, AccB7I, AccBSI, AclNI,
  AfeI, AflII, AflIII, AgeI, AlwNI, AocI, Aor51HI,
  ApaI, AscI, AseI, AsnI, Asp700I, AspI, AtsI,
  AviII, AvrII, BamHI, BbrPI, BbuI, BclI, BfrI,
  BglI, BglII, BlnI, BlpI, Bpu1102I, Bpu14I, BsaI,
  BsaMI, Bse118I, Bse21I, BsePI, BseRI, BsgI, BsiI,
  BsmI, Bsp119I, Bsp120I, Bsp1720I, Bsp19I,
  Bsp68I, BspCI, BspLU11I, BspTI, BsrBI, BsrFI,
  BssAI, BssHII, BssSI, Bst98I, BstBI, BstD102I,
  BstDSI, BstEII, BstI, BstPI, BstSNI, Bsu36I,
  CciNI, CelII, Cfr10I, Cfr42I, Cfr9I, CpoI,
  Csp45I, CspI, CvnI, DraI, DrdI, DsaI, Eam1104I,
  EarI, Ecl136II, Eco105I, Eco147I, Eco255I,
  Eco31I, Eco47III, Eco57I, Eco72I, Eco81I,
  Eco91I, EcoICRI, EcoNI, EcoO65I, EcoT22I,
  Esp1396I, FbaI, FseI, FspI, HindIII, HpaI,
  Ksp22I, Ksp632I, KspI, LspI, MfeI, MluI,
  Mph1103I, MroNI, MspCI, MunI, Mva1269I, NaeI,
  NcoI, NgoAIV, NgoMI, NheI, NotI, NruI, NsiI,
  NspI, NspV, PacI, PaeI, PaeR7I, PflMI, PinAI,
  Ple19I, PmaCI, Pme55I, PmeI, PmlI, Ppu10I,
  PshBI, Psp124BI, PspAI, PspALI, PspEI, PspOMI,
  PstI, PstNHI, PvuI, RsrII, SacI, SacII, SalI,
  SapI, SbfI, ScaI, SfiI, Sfr274I, Sfr303I, SfuI,
  SgfI, SgrAI, SmaI, SmiI, SnaBI, SpeI, SphI,
  SrfI, Sse8387I, SseBI, SstI, SstII, StuI, SwaI,
  Tth111I, Van91I, Vha464I, VspI, XbaI, XhoI,
  XmaI, XmnI, Zsp2I

38
As a conclusion

• BamH1 and Xho1 are selected for the restriction
  enzymes because they do not cut inside the gene
  interest.

39
BLAST Analysis

• To ensure that the gene sequence that we choose
  is exact gene sequence of the gene interest, dexA
  gene for dextranase.
• To compare the similarity of our gene sequence to
  other gene.

40
BLAST ANALYSIS (cont.)

• there are two gene from the Penicillium
  minioluteum (clone pUDEX) dextranase (Dex) gene
  and Penicillium minioluteum isoform (Dex 2) gene
  have similarity with Penicillium funiculosum dexA
  gene for dextranase but not 100 of similarity.

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42
Multiple DNA sequence alignment

• PURPOSE - to compare the other genes which are
  similar to our gene of interest.
• We can know the different part of our gene if
  compare to other genes which is similar to our
  gene of interest.

43
Multiple DNA sequence alignment (cont.)

• Penicillium minioluteum (clone pUDEX) dextranase
  (Dex) gene and Penicillium minioluteum isoform
  (Dex 2) gene.
• Why we chose these genes?
• Origin from the same genus (Penicillium)
• the gene sequence are almost similar to the our
  gene of interest.

44
Multiple DNA sequence alignment (cont.)
45
Multiple DNA sequence alignment (cont.)
46
Choosing the suitable vector

• pETBlue-2 vector
• REASON
• The presence of the desired enzyme cleavage sites
  in the multiple cloning sites (MCS)
• Allows T7 lac promoter based expression of target
  genes - blue/white screening.

47
Choosing the suitable vector (cont.)

• Features an expanded multiple cloning site (MCS)
  and optional c-terminal HSV.Tag and His.Tag
  sequences - protein purification
• has the following elements T7 promoter,f1 origin
  of replication, lac Z gene,pUC origin,E. coli
  promoter,Lac operator region 3 to the T7
  promoter,Ampicillin resistance marker and EK site

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49
DESIGN PRIMER FOR PCR AMPLIFICATION

• Primers
• forward and reverse primer
• produce end product of PCR that contain two
  restrictions recognize site (based on pETBlue-2)

50

• length 26 bp
• - 6 nucleotides for both primer is a DNA
  sequence which will recognize by
  restriction enzyme BamH1 and Xho1
• - 20 nucleotides are based on the sequence at
  the 5 end to 3 end.

51
Forward Primer 5atggatccatggccacaatgctaaagct3 A
7, T 4, G 4, C 5 GC content 45   Tm
(AT) 2 (GC) 4 (74)2 (45)4
22 36 58C   Ta 58C-2C 56C
Reverse Primer 5atctcgagtcacgcgctctgagagctgg3 A
3, T 4, G 7, C 6 GC content 65   Tm
(AT) 2 (GC) 4 (34)2 (76)4
14 52 66C   Ta 66C-2C 64C
52
Cutting site
53
END PRODUCT OF PCR
5 ggatccatggccacaatgctaaactctcagagcgcgtgact
cgag 3
3 cctaggtaccggtgttacgatttga
gagtctcgcgcactgagctc 5
Gene of Interest 7 1857 bp
Xho l recognize site
Bam Hl recognize site
54
POLYMERASE CHAIN REACTION

• 3 steps
• 1. Denaturation
• 2. Annealing
• 3. Extension / replication
• 1. DENATURATION
• - at 95 C
• - double strand of DNA melts ? open to
  single stranded DNA

55

• 2. ANNEALING
• - primers bind to the single strand DNA
  (complementary)
• - hydrogen bonds form between the primer and
  the DNA strand
• - double helix structure is re-created
• - temperature 56 C
• 3. EXTENSION
• - primer which anneal at the single strand DNA
  will starts the process of multiplying the
  amount of DNA
• - primer that are on position with no exact
  match get loose again and dont give an
  extension of the fragment
• - temperature around 72 C
• - used of Taq DNA polymerase

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PROTOCOL FOR PCR

•   Tube S (µl) Tube C
  (µl)
• Master mix 40 40
• Forward primer 2.5 2.5
• Reverse primer 2.5 2.5
• Template DNA(gene of interest) 5 0
• Sterile deionised distilled water 0
  5
• Total 50 50

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Separation of product by agarose gel
electrophoresis
58
LIGATION

• Then, the restricted dexA-linker complex will be
  ligated into the restricted pETBlue-2 by using
  the ligation enzyme which is T4 DNA ligase.
• The process of DNA ligation involves creating a
  phosphodiester bond between the 3 hydroxyl of
  one nucleotide and the 5 phosphate of another.
• The T4 DNA ligase requires ATP for its activity.

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BamH1
Xho1
DexA gene
Recombinant
60
TRANSFORM OF RECOMBINANT INTO E.COLI (NovaBlue
strain)
61
TRANSFORM OF RECOMBINANT INTO E.COLI

• Transformation is the uptake of recombinant
  plasmid DNA into host (E. coli).
• Firstly we make the bacterial cells competent and
  then introduce recombinant plasmid with dexA
  gene.
• This can be done by treating the cells either
  with calcium chloride (CaCl2) method or by means
  of electroporation.

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Diagram shown the transformation using the
calcium chloride method
63
VERIFICATION OF SUCCESSFUL TRANSFORMATION

• Grow the E. coli onto LB ampicillin plate.
• The plasmid consists of the ampicillin resistance
  gene that will help to protect the E. coli from
  the antibiotic ampicillin.
• If there are colonies present in the ampicillin
  plate, it means the transformation of the plasmid
  into E. coli is successful.

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Blue White colonies screening

• pETBLUE-2 has MCS where located on the lac z
  gene.
• If the DexA gene insert to MCS of Vector
  pETBlue-2 successfully, the lac z gene will
  inactivate.
• This is called Insertional Inactivation of
  beta-galactosidase.
• Non-functional beta-galactosidase will produce.
• can not breakdown of X-gal to a product that is
  colored deep blue.
• E.coli carrying this plasmid will form white
  colony.

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If no DexA gene insert into MCS of vector
pETBlue-2, beta-galactosidase will produce.
This will break down X-gal to a product that is
colored deep blue. E.coli carrying this plasmid
will form blue colony.
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Secondary Screening of Blue White Colonies

• Lead to up to 40 of false-positives for the
  insertion.
• White colonies on X-Gal do not always show true
  colors.
• to eliminate the false positive in blue white
  screening.
• Can get positive insert clones more accurately .
• The methodology same as blue white screening.
• true positive can take for further screening.

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Restriction Enzyme Cleavage Pattern

• True positive white colony grow in large amount
  in agar plate containing Ampicillin antibiotic.
• The colonies treat with two restriction enzymes
  (BamH l Xho l) before run agarose gel
  electrophoresis

68
PCR Screening

• gene inserted vector (pETBlue-2) extracted out
  from the true positive colony and doing the PCR
  screening.
• The end product of PCR will treat with
  restriction enzymes before load into agarose gel
  for running the gel electrophoresis.

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Expression of the Recombinant Protein

• Principle using the promoter sites that helps
  to express a foreign protein in the host cells.
• The recombinant E. coli are let to be grown up
  on the nutrient agar plate
• Then the bacteria are treated with IPTG
• IPTG molecules are the inducer that increase the
  production of recombinant protein
• T7 promoter then is activated to start the
  transcription and translation of dexA gene into
  enzymes dextranase

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Protein Purification

• Nickel Metal Affinity Chromatography Presence
  of His-tag inside the vector
• Principle separate the protein base on the
  isoelectrical charge and the biological activity
  of the proteins
• Before the protein purification steps, the
  E.coli need to be lysed to release the abundant
  of proteins.

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Purification of the His-tag recombinant protein
Recombinant protein with His tag
Pass it through a Nickel affinity column
Wash the unbound protein or protein without the
His tag with buffer
Elute the recombinant protein with His tag by
either lowering the pH or using imidazole
His tag protein is treated with the specific
protease to cleave off the His tag
The recombinant protein is freed of the His tag
peptide by running it over the metal-chelate
column again
72
Diagram shown the steps involve in purification
of protein
73
Why SDS-PAGE ?

• After the protein purification, the protein
  that obtained must be analyzed to make sure the
  protein we got is pure protein (Dextranase).

74
Why SDS-PAGE ?

• After SDS PAGE process,only one band protein
  appears on the gel,
• 67.7 kDa
• This estimation of the molecular weight of the
  Dextranase can be done by comparing with the
  known molecular weight marker.
• An Rf graph can be drawn using the known
  molecular weight marker and read the molecular
  weight of Dextranase through the graph.
• If more than one band appears on the gel, its
  mean that there is a mixture of the protein that
  we obtained and the protein is not pure.

75
Test the functioning of enzyme

• After we obtain the pure protein, we have to do
  tests to make sure that the protein that we
  cloned out is functioning
• There are several test that to assay the enzymes
• -Degradation of glucans with hydrolytic enzymes
• -Stability in commercial mouthwashes
• -Temperature stability

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Test the functioning of enzyme

• 1 ml of dextranase was added to 4 ml commercial
  mouthwash without ethanol and 4 ml of the same
  product with ethanol.
• The tube keep in room temperature.
• Aliquots were removed at different times and the
  residual activity was determined in standard
  conditions using maltose as a substrate.

77
Test the functioning of enzyme

• Expected result
• Dextranase exhibited good stability in
  mouthwash without ethanol at pH 5.7 and at room
  temperature and they kept 77 of their initial
  activity after 4 month.
• Under same condition in mouthwash containing
  ethanol dexranase only showed only 23 of initial
  activity

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79
Commercial Values

• With declining caries experience, increasing
  number of people retaining their teeth new
  emphasis on cosmetically attractive dentitions,
  there is an increasing public awareness of the
  value of personal oral hygiene.
• Potential use of dextranase
• decrease the dietary substances of the
  microorganisms
• promote buffer to neutralize the plaque
  acids
• provides antibacterial substances which
  inhibit dental plaque formation

80
Commercial Values

• We plan to combine the use of dextranase along
  with the commercial product that already exist in
  the market such
• chewing gum
• dental products like toothpaste and mouthwash

81
Conclusion

• The dexA gene is the gene that produces the
  dextranase, a type of protein that helps to
  degrade the dental plaque.
• Dextranase have a potential market expansion,
  especially with therapeutic or cosmetic
  attributes

82
REFERENCES

• http//www.ncbi.nlm.nih.gov (14.08.2003)
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  3_www.cgi (19.08.2003)
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83
REFERENCES

• Invitrogene 1998 Catalog
• Novagen 2002 Catalog
• M. Marotta, A. Martino, A. De Rosa, E. Farina, M.
  Cartenì and M. De Rosa. Degradation of dental
  plaque glucans and prevention of glucan formation
  using commercial enzymes. ScienceDirect. Volume
  38, Issue 1 , September 2002 , Pages 101-108.
• Robert J. Brooker, Genetics Analysis and
  Principle, Addison-Wesley, 1999.
• Smith and Wood, Molecular Biology and
  Biotechnology, Chapman Hall, 1991.
• T. A. Brown, Gene Cloning An Introduction,
  Chapman Hall, 2nd edition.
• http//www.woodrow.org/teachers/bi/2000/Doggy_DNA/
  background_for_polymerase_chai.html
• http//allserv.rug.ac.be/avierstr/principles/pcr.
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