Analysis of Putative Sago Palm Cysteine Protease cDNA Open Reading Frame

JONG THIN THIN

Bachelor of Science with Honours

(Resource Biotechnology)

2013

Faculty of Resource Science and Technology

Analysis of Putative Sago Palm Cysteine Protease cDNA Open Reading Frame

Jong Thin Thin (26533)

A final project report submitted in partial fulfilment of the Final Year Project II

(STF 3015) Course

Supervisor: Associate Professor Dr. Hairul Azman @ Amir Hamzah Bin Roslan

Co-supervisor: Ms. Norafila Humrawali

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

2013

II

DECLARATION

I declare that the thesis entitled “Analysis of Putative Sago Palm Cysteine Protease cDNA

Open Reading Frame” hereby submitted for the STF 3015 Final Year Project 2 at the

University Malaysia Sarawak (UNIMAS) is my own work and have not been previously

submitted by me at another University for any degree. I cede copyright of the thesis in

favor of the University Malaysia Sarawak (UNIMAS). Formulations and ideas taken from

other sources are cited as such. This work has not been published.

___________________

Jong Thin Thin (26533)

Biotechnology Resource

Faculty of Resource Science and Technology

University Malaysia Sarawak

I

ACKNOWLEDGEMENT

Upon completion of my final year project, I am contented and enjoyed the process of doing

the project. I did gain a lot of informative and technical knowledge by doing this

meaningful academic research study. Apart from the efforts of myself, the success of this

project depends largely on the encouragement and guidelines of many others. This research

project would not have been possible without the support of many of them. Therefore, I

take this opportunity to express my deepest gratitude for those who have been instrumental

in the successful completion of this project.

First and foremost, I would like to offer my special thanks to my supervisor, Associate

Professor Dr. Hairul Azman @ Amir Hamzah Roslan, for giving me an opportunity to

work on this project under his guidance. His valuable and constructive suggestions during

the planning and development of this research work are very vital for the success of this

project. His willingness to give his time so generously has been very much appreciated. I

also want to deliver special thanks to Ms. Norafila Humrawali for her guidance.

Besides that, I would also like to acknowledge the help provided by the master students of

Dr. Hairul, for the guidance, and sharing experiences and knowledge with me throughout

my project. Special thanks are also extended to my coursemates for sharing the literature

and invaluable assistance.

Finally, I wish to express my love and gratitude to my beloved families; for their

understanding, support, encouragement, and endless love, through the duration of my

studies.

III

TABLE OF CONTENTS

ACKNOWLEDGEMENT ........................................................................................................ I

DECLARATION ...................................................................................................................... II

TABLE OF CONTENTS ....................................................................................................... III

LIST OF ABBREVIATIONS .................................................................................................. V

LIST OF TABLES .................................................................................................................. VI

LIST OF FIGURES ............................................................................................................... VII

ABSTRACT ............................................................................................................................ IX

1.0 INTRODUCTION ............................................................................................................... 1

2.0 LITERATURE REVIEW ................................................................................................... 3

2.1 Cysteine Protease ......................................................................................................... 3

2.2 Cloning Vector ............................................................................................................. 5

2.2.1 The pET-41a(+) Expression System ..................................................................... 6

2.3 Restriction Enzyme ...................................................................................................... 8

2.4 DNA Ligase ............................................................................................................... 10

2.5 Bacterial Transformation Process in Plant ................................................................. 11

3.0 Materials and Method ....................................................................................................... 14

3.1 Media Preparation: Luria Broth (LB), Luria Agar (LA) and Calcium Chloride

(CaCl2) ............................................................................................................................. 14

3.2 Transformation of Escherichia. coli XL1 Blue with pBluescript Plasmid ................ 14

3.3 Isolation of Double-stranded pBluescript Plasmid DNA from E. coli XL1 Blue ..... 16

3.4 cDNA Insert Preparation............................................................................................ 17

3.5 The pET-41a(+) Vector Preparation .......................................................................... 19

3.6 DNA Extraction from Agarose Gel ........................................................................... 19

3.7 Restriction Endonuclease Digestion of DNA ............................................................ 20

3.8 DNA Ligation Process ............................................................................................... 21

3.9 Transformation of E. coli XL1 Blue with pET-41a(+)-msCPR cDNA ORF ............ 21

3.10 Screening Clone ....................................................................................................... 22

4.0 RESULTS ........................................................................................................................... 24

4.1 Transformation of E. coli XL1 Blue with pBluescript Plasmid which Containing

msCPR cDNA ORF ......................................................................................................... 24

IV

4.2 Isolation of Double-stranded pBluescript Plasmid DNA from E. coli XL1 Blue ..... 25

4.3 cDNA Insert Preparation............................................................................................ 26

4.4 The pET-41a(+) vector preparation ........................................................................... 27

4.5 Retriction digestion of DNA ...................................................................................... 28

4.6 Transformation of E. coli XL1 Blue with pET-41a(+)-msCPR cDNA ORF ............ 29

4.7 Screening Clone ......................................................................................................... 30

4.7.1 Colony PCR......................................................................................................... 30

4.7.2 Restriction Fragment Analysis ............................................................................ 31

4.7.3 DNA Sequencing ................................................................................................. 32

5.0 DISCUSSION ..................................................................................................................... 33

6.0 CONCLUSION .................................................................................................................. 43

7.0 REFERENCES .................................................................................................................. 44

V

LIST OF ABBREVIATIONS

msCPR Metroxylon sagu cysteine protease

DNA Deoxyribonucleic acid

cDNA Complementary deoxyribonucleic acid

IPTG Isopropyl-b-d-thiogalactopyranoside

LB Luria broth

LA Luria agar

CaCl2 Calcium chloride

NaCl Sodium chloride

Rpm Revolution per minute

Rcf Relative centrifugal force

OD 600 Optical density 600

°C Degree celcius

ml milliliter

Mm millimolar

% Percent

l Microliter

g Gram

PCR Polymerase chain reaction

EtBr Ethidium bromide

kb Kilobase pair

bps Base pairs

VI

LIST OF TABLES

Table 1: Recipe of master mix. ................................................................................................. 17

Table 2: The thermal cycler condition of the PCR process. ..................................................... 18

Table 3: Reagents needed for restriction endonuclease digestion of DNA. ............................. 20

Table 4: Reagents needed for DNA ligation process. ............................................................... 21

Table 5: Reagents needed for restriction endonuclease digestion of DNA. ............................. 22

VII

LIST OF FIGURES

Figure 1: Action of catalysis mechanism. ................................................................................... 3

Figure 2: The map of pET-41a(+) vector. .................................................................................. 7

Figure 3: The catalytic action of DNA ligase. (Adapted from: Sreedhara et al., 2004). .......... 10

Figure 4: Plant transformation. ................................................................................................. 12

Figure 5: Schematic diagram of the micro-shock wave devices. ............................................. 13

Figure 6: An agar plate that grown with colonies of E. coli XL1 Blue containing

pBluescript-msCPR cDNA ORF. .............................................................................................. 24

Figure 7: The photographic image of an agarose gel electrophoresis analysis of

pBluescript plasmid containing msCPR cDNA ORF which extracted from E. coli XL1

Blue. ........................................................................................................................................... 25

Figure 8: The photographic image of an agarose gel electrophoresis analysis of PCR

product of pBluescript plasmid which extracted from E. coli XL1Blue. .................................. 26

Figure 9: The photographic image of an agarose gel electrophoresis image of pET-41a(+)

plasmid....................................................................................................................................... 27

Figure 10: The photographic image of an agarose gel electrophoresis image of pET-41a(+)

plasmid and msCPR cDNA ORF that had been linearized with restriction enzyme, Nde I

and Not I. ................................................................................................................................... 28

Figure 11: An agar plate that grown with colonies of E. coli XL1 Blue containing pET-

41a(+)-msCPR cDNA ORF. ...................................................................................................... 29

Figure 12: The photographic image of an agarose gel electrophoresis analysis of PCR

product of pET-41a(+)-msCPR cDNA ORF plasmid which extracted from E. coli XL1Blue.

................................................................................................................................................... 30

Figure 13: The photographic image of an agarose gel electrophoresis analysis of the

comparison of pET-41a(+)-msCPR cDNA ORF plasmid that had been linearized with

VIII

restriction enzyme, Nde I and Not I with the uncut pET-41a(+) plasmid and pET-41a(+)-

msCPR cDNA ORF plasmid. .................................................................................................... 31

Figure 14: The nicked-open circular and supercoiled plasmid DNA. (Adopted from

VALUE, 2013). ......................................................................................................................... 37

Figure 15: The part of chromatogram of the data. .................................................................... 41

Figure 16: The part of chromatographic fluorescence data. ..................................................... 41

IX

Analysis of Putative Sago Palm Cysteine Protease cDNA Open Reading Frame

JONG THIN THIN

Resource Biotechnology

Department of Molecular Biology

Faculty of Resource Science and Technology

Universiti Malaysia Sarawak

ABSTRACT

Cysteine protease is one of the types of proteolytic enzyme that present in Metroxylon sagu and has many

roles in plant cells physiology and development process. In this study, the objective is to clone a putative

cysteine protease gene derived from M. sagu into an expression vector. Firstly, the msCPR cDNA ORF

which has been isolated from sago palm, was propagated in pBluescript plasmid. The clone was analyzed via

restriction enzyme and PCR. The second objective is to clone the msCPR cDNA ORF into an expression

vector, pET-41a(+). The last objective is to determine the correct insertion of the coding fragment of pET-

41a(+) which containing msCPR cDNA ORF through DNA sequencing reaction. A pBluescript vector

containing the putative msCPR was checked via PCR and produced a fragment with the size of 750 bps. Then,

the pET-41a(+) vector and msCPR cDNA ORF was successfully digested with Nde I and Not I restriction

enzyme and subsequently ligated together. However, the determination of the correct insertion of the coding

fragment of pET-41a(+)-msCPR cDNA ORF through DNA sequencing reaction was failed to be conducted.

Key words: Cysteine protease, pET-41a(+), restriction enzyme, ligation, DNA sequencing

ABSTRAK

Protease Cysteine adalah salah satu jenis enzim proteolitik yang terdapat dalam Metroxylon sagu yang

berperanan dalam sel-sel tumbuhan fisiologi dan proses pembangunan. Dalam kajian ini, objektif adalah

untuk mengklonkan putatif gen cysteine protease yang diperolehi daripada M. sagu ke dalam ekspresi vektor.

Pertama kali, msCPR cDNA ORF yang telah diasingkan daripada sagu telah dicantumkan dalam plasmid

pBluescript. Klon telah dianalisasikan melalui enzim retriksi dan PCR. Objektif kedua adalah untuk

mengklonkan msCPR cDNA ORF ke dalam vektor ekspresi, pET-41a(+). Objektif terakhir adalah untuk

menentukan kemasukan serpihan kod pET-41a(+) yang mengandungi msCPR cDNA ORF yang betul melalui

penjujukan DNA tindak balas. Vektor pBluescript yang mengandungi putatif msCPR telah diperiksa melalui

PCR dan menghasilkan serpihan yang bersaiz 750 bps. Kemudian, pET-41a(+) vektor dan msCPR cDNA

ORF telah berjaya dipotong dengan enzim retriksi Nde I dan Not I dan sterusnya pemasangannya bersama-

sama. Tetapi, pengenalpastian serpihan kod pET-41a(+)-msCPR cDNA ORF yang betul melalui penjujukan

DNA tindak balas telah gagal dijalankan.

Kata kunci: Protease cysteine, pET-41a(+), enzim retriksi, ligasi, penjujukan DNA

1

1.0 INTRODUCTION

Cysteine protease is one of the types of proteolytic enzyme that present in most of the

organisms which include animals, plants, some bacteria and viruses (Joo et al., 2007).

Cysteine protease carries out its function via catalytic mechanism whereby mediated

cleavage of a peptide bonds of a polypeptide. In plants, cysteine protease gene was shown

to be expressed during growth and development stage (Matarasso et al., 2006). Besides

that, Solomon and his colleagues (1999) investigated that cysteine protease plays an

essential role in the regulation of plant senescence and in the induction of programmed cell

death under stressed condition. Therefore, this enzyme also serves as a messenger in the

signaling pathway in order to response to the biotic and abiotic stress (Grudkowska &

Zagdariska, 2004). In addition, Mohan et al. (2008) had reported that cysteine protease

function as a defense protein by possessing a remarkable toxicity against insects and also

pathogens.

Cysteine protease comprise of more than 40 families which are further sub-

classified into at least six superfamilies (Grudkowska & Zagdariska, 2004). Grudkowska

and Zagdariska (2004) stated that most of the cysteine proteases that present in plant are

papain and legumain families. According to Peng et al. (2008), papain from the latex of

Carica papaya is the most widely studied cysteine protease in plant. This gene can also be

found in sago palm (Metroxylon sagu). Through the analysis of EST, Wee and Hairul

(2011) had found that cysteine protease in M. sagu is expressed when the plants encounter

stresses, such as pathogen attack. M. sagu is a very versatile and physically hard

monocotyledonous plant (Wee & Hairul, 2011). It is economically important and now

grown commercially in Malaysia for the starch production and also ethanol fuel production

as well as in food industry (McClatchey et al., 2006). Recently, there is an occurrence of

new competition in the production of biofuel and food due to the exhaustion of fossil

2

energy and the increment of world population (Ehara, 2012). M. sagu becomes a new

source needed to solve this problem because of its potential in the starch production (Ehara,

2012). In addition, it can tolerate salinity, prolonged flooding, and acidic peat soils

(McClatchey et al., 2006). Due to its economic value and adaptability towards such

environment, cysteine protease gene is isolated and the cloned into an expression system

for this study.

The discovery of isolation the msCPR cDNA ORF from sago palm prompted the

work to express the cysteine protease gene. Therefore, a vector containing msCPR cDNA

ORF was be constructed. The pET-41a(+) was selected in order to clone the msCPR cDNA

ORF because it has high level expression of recombinant protein and very suitable for

cloning. Before the cloning process, restriction enzyme was used to digest the msCPR

cDNA ORF and pET-41a(+) vector in order to produce sticky end which is complementary

with each other. Then, the positive clones were identified and followed by the

transformation process. DNA sequencing was conducted in order to ensure the correct

insertion of the coding fragment of pET-41a(+)-msCPR cDNA ORF.

In this study, there are three objectives to be taken into account. The first objective

is to obtain the msCPR cDNA ORF which has been isolated from sago palm from a

selected vector, called pBluescript. The second objective is to clone the msCPR cDNA

ORF which has been isolated from sago palm into a selected expression vector, pET-

41a(+). The last objective of this study is to determine the correct insertion of the coding

fragment of pET-41a(+) which containing msCPR cDNA ORF through DNA sequencing

reaction.

3

2.0 LITERATURE REVIEW

2.1 Cysteine Protease

Cysteine protease is a group of intracellular protease enzyme that plays a key role to

catalyse the cleavage of peptide bonds in a protein. This enzyme cleaves the bonds

between the amino acids in a polypeptide chain by hydrolysis. According to Grudkowska

and Zagdariska (2004), the protease enzymes can be classified into two types:

endopeptidase and exopeptidase. Endopeptidase cleave the peptide bonds on the interior

part of the polypeptide chain which is based on their active side residue; while,

exopeptidase cleave on the end part of the polypeptide chain which is based on their

substrate specificity (Grudkowska & Zagdariska, 2004). According to protease

classification, cysteine protease is an endopeptidase with a cysteine residue in their active

site. The action of catalysis mechanism is shown as follows:

Figure 1: Action of catalysis mechanism.

Cysteine protease consists of more than 40 families and sub-classified into at least

six clans (Grudkowska & Zagdariska, 2004). Among the protease enzyme, cysteine

protease is the first enzyme studied; and Schaller (2004) believed that this enzyme plays an

important role in plants. Papain is the first cysteine protease that was discovered in the

latex and fruit of Carica papaya, and the most extensively studied protease (Schaller, 2004).

4

Grudkowska and Zagdariska (2004) stated that the genome of Arabidopsis thaliana

contains the gene which codes for 32 types of papain-like cysteine protease.

From the previous study, they found out that among 42 proteinases, there were 27

cysteine proteases took part in barley seed germination. In maize, 90% of the degradation

activity was controlled by cysteine protease (Grudkowska & Zagdariska, 2004). Therefore,

this indicates that cysteine protease plays a vital role in protein degradation and

mobilization. Besides that, Solomon (1999) stated that programmed cell death is occurred

as the cysteine protease is being induced by environmental-stress. Grudkowska and his

coworkers (2004) had proved that wheat can tolerate frost conditions because of the

activity of cysteine protease gene in their genome. In addition, the plant cysteine proteases

also serve as a defense mechanism by possessing toxicity against insect larvae such as for

papain from papaya (Carica papaya) that has been proved to act in the defense mechanism

against insect larvae.

A study conducted by Nieuwenhuizen et al. (2012) stipulated that cysteine protease

in the kiwifruit has an ability in the application of degradation of gelatin. Cysteine protease

enzyme also plays an important role in the medical field to treat allergenicity

(Nieuwenhuizen et al., 2012). This indicates the great functions of the cysteine protease in

the plant.

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2.2 Cloning Vector

Vector is known as a small piece of DNA molecule which serves as an agent to carry a

gene from the donor to the host cell. In the era of biotechnology, vector is widely used for

DNA cloning in order to produce genetically modified organisms. As stated by Levine

(2007), cloning was first invented at the twentieth century and this technology was

successfully created a cloned sheep in 1996. This cloned sheep project had prompted the

more advanced work of producing transgenic organisms. On the other hand, Goldberg

(2001) stipulated that plant cloning era was established at the end of the 1970s after the

principles of plant genome organization and gene regulation were known by the scientists.

John Bedbrook and his colleagues had proved that plant DNA can be cloned and replicated

in bacteria as other organisms DNA (Goldberg, 2001). They had successfully cloned the

ribosomal DNA and telomeric repeated sequences from wheat into the vector system by

using the same enzymes which are similar to all other organisms used (Goldberg, 2001).

After that, many plant genome libraries had been constructed in the early 1980s by

using the vector cloning method. Those constructed libraries make the plant genome

available to scientists all over the world. Besides that, Goldberg (2001) had mentioned that

plant cDNA libraries were also constructed in order to study the plant gene expression at

the level of transcription. At the same time, developmental, metabolic, as well as

biochemical process of plant can be studied through the discoveries of plant cDNAs.

There are many researches such as the establishment of Arabidopsis and rice

genome projects had been successfully discovered by using this technology (Goldberg,

2001). Many sequencing projects which had helped to uncover tens of thousands of mRNA

in a wide range of plants by using the technology of expressed sequence tag (Goldberg,

2001). In addition, many transgenic plants have been created with invention of the vector

cloning technique. In 2000, golden rice had produced by modified the metabolic pathway

6

and subsequently introduced into the endosperm of rice, so that high yield of beta carotene

can be obtained and vitamin A can be produced in the large quantities (Key et al., n.d.).

In the modern plant biology and biotechnology era, many new discoveries and

robust development have been introduced by the scientists. New vector system has been

introduced in order to facilitate the simple cloning strategies and also high efficiency in the

gene expression. A set of ligation-independent cloning vectors has been launched for the

functional studies in plants (Rybel et al., 2011). This type of vector is very effective

because moderately high throughput fashion can be applied in verifying protein

localizations. Precise cloning is allowed by using ligation-independent cloning vectors

(Rybel et al., 2011). The advancement in this field prompted the work in the field of plant

molecular study.

2.2.1 The pET-41a(+) Expression System

The pET expression system is widely used in the field of cloning or recombinant DNA due

to its high efficiency in transcription and translation (Agilent Technologies, 2012). Protein

expression of pET system can be induced by using IPTG. The pET expression vectors are

originally derived from pBR322 plasmid, are engineered in order to establish a system with

high efficiency in transcription and also translation (Agilent Technologies, 2012). For the

pET-41 series, it is specifically designed for the high level of protein expression through

the popular GST fusion tag (Novagen, n.d.).

7

Figure 2: The map of pET-41a(+) vector.

. (Adapted from: Novagen, n.d.).

8

2.3 Restriction Enzyme

Restriction enzyme is an enzyme which functions to cleave the DNA at the recognized

specific site; often four, six or eight base pairs long, and subsequently to produce

fragments. The discovery of restriction enzyme is a great contribution in the field of

biotechnology as it serves as an important tool in the DNA recombinant technology

toolbox and makes genetic engineering possible. According to Nwankwo and Abalaka

(2011), restriction enzyme is found in the bacteria and some archaea bacteria which serve

as a defense mechanism to against invading viruses.

According to Nwankwo and Abalaka (2011), restriction enzyme was discovered by

a Swiss microbiologist, Werner Arber who received 1978 Nobel Prize in Physiology and

Medicine. Werner Arber and his colleagues found out that bacterial cell able to self-

defense against the foreign DNA through the study of phenomenon of host-controlled

restriction of bacteriophages (Pray, 2008). During that study, they observed that phage

particles are often unable to grow well and infect other strains of the same bacterial species

as they can grow well and efficiently infect one strain of bacteria (Pray, 2008). Besides that,

phage particles show opposite pattern which are able to grow well and infect a second

strain of bacterial species efficiently as they grow poorly in the original strain (Pray, 2008).

Furthermore, the study also showed that phage particles which able to grow and infect host

cells efficiently possess a DNA modification by the addition of methyl groups to the

adenine or cysteine bases, while the phage particles which only show poorly infection to

their host cells that does not have DNA methylation pattern (Pray, 2008). Pray (2008)

highlighted that phage particles were said to be restricted by their host as they only grow

and infect poorly to their host cells.

There are different types of restriction enzymes have been created. Restriction

enzymes are categorized into four categories, which are Type I, Type II, Type III, and also

9

artificial restriction enzymes (Nwankwo & Abalaka, 2011). Different restriction enzymes

cleave at different sequence of nucleotides and produce double-stranded cut in the DNA

which are differ in sequence, length, and strand orientation of a sticky end and also blunt

end (Nwankwo & Abalaka, 2011). As postulated by Pray (2008), “Type I restriction

enzyme recognize specific DNA sequences but cut at the random sites that can be as far as

1000 base pairs away from the recognition site; Type II recognize and cut directly within

the recognition site; and Type III recognize specific sequences but cut at different specific

location that is usually within about 25 base pairs of the recognition site”. Whereas,

artificial restriction enzymes are created by fusing engineered DNA binding domain to a

nuclease domain; so that, it is able to bind to desired DNA sequences (Nwankwo &

Abalaka, 2011). The most commonly used artificial restriction enzymes especially used in

genetic engineering applications and also standard gene cloning applications are zinc finger

nucleases (Nwankwo & Abalaka, 2011).

Nowadays, restriction enzymes play an important in genetic engineering as it helps

to insert the desired genes that have been cut into the plasmid vectors for gene cloning

purposes (Nwankwo & Abalaka, 2011). Besides that, it also widely used in polymerization

chain reaction (PCR) DNA-base manipulation in-vitro, molecular husbandry, southern

blotting analysis genetic engineering, restriction fragment length polymorphism (RFLP)

analysis, DNA mapping, and many others application in biotechnology (Nwankwo &

Abalaka, 2011).

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2.4 DNA Ligase

DNA ligase is a kind of enzyme that widely used in catalyzing the formation of

phosphodiester bond to join the 5’ phosphate of one strand of DNA to the 3’ hydroxyl of

another strand of DNA. ATP, as the energy source is required for this catalytic reaction to

carry out efficiently. According to Ellenberger and Tomkinson (2008), DNA ligases play a

vital role in DNA replication, repair, and also recombination. The catalytic action of DNA

ligase is illustrated in Figure 3.

Figure 3: The catalytic action of DNA ligase. (Adapted from: Sreedhara et al., 2004).

DNA ligase has become an important tool in the molecular biology research for

genetic engineering purposes. It helps to join the desired gene fragment into the selected

plasmid vector in order to produce a genetically modified gene products. T4 DNA ligase is

the most widely used DNA ligase in the field of molecular biology. T4 DNA ligase is a

single polypeptide which derived from T4 bacteriophage (VALUE, 2013).

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2.5 Bacterial Transformation Process in Plant

Transformation is processes that only can be carried out by bacteria to uptake free DNA

actively and subsequently integrate the genetic information into the cell (Lorenz &

Wackernagel, 1994). As highlighted by Lorenz and Wackernagel (1994), transformation

was first invented by Griffith who had discovered the transformation in Streptococcus

pneumonia in 1928. Avery and his coworkers had demonstrated that “DNA is a

transforming principle and came out some ideas about bacteria might be favored subjects

for genetic investigation and eventually for technological application of molecular genetics

science” (Lederberg, 1994).

Through this idea, bacterial transformation had become very popular in many

studies in the past. The bacteria have been widely investigated include Pneumococcus,

Haemophilus influenza, Bacillus subtilis, and some others bacteria (Goldberg, 2001).

Previously, some scientists thought that Escherichia coli were not responding to

transformation. However, Cohen and his coworkers (1972) had claim that E. coli can be

transformed with the treatment of calcium chloride. The discovery of E. coli

transformation had prompted the work of discovery of artificially-induced competence in E.

coli which is simpler to carry out for molecular cloning (Cohen et al., 1972). Froger and

Hall (2007) had reported that transformation of plasmid DNA into E. coli can be done by

using the heat shock method. Besides that, Singh et al. (2010) found out that

transformation efficiency was ~24 fold higher when the calcium chloride treated cells were

further incubated on ice for 10 min after heat shock when compared to no heat shock and

only heat shock treatment. Transformation is now extensively used in molecular biology

field in order to produce transgenic organism. The illustration of plant transformation is as

follows:

12

Figure 4: Plant transformation.

(Adapted from: Genetically Engineered Crops, n.d.).

Furthermore, many researchers had started focus on the study of Agrobacterium

tumefaciens and its causative agent at around 90 years ago (Riva et. al., 1998). They found

out that A. tumefaciens has an ability to integrate its DNA of the tumor-inducing plasmid

into the nucleus of the host cells. Dohbal et al. (2010) used neomycin phosphotransferase

II (npt II) and glucuronidase (GUS) as reporter genes in order to study the transformation

efficiency of using A. tumefaciens. According to Dohbal et al. (2010), A. tumefaciens is the

most used method for the plant transformation process whereby help in delivery of gene of

interest into a host nuclear genome with the high transformation frequency.

Recently, there is a new invention for bacterial transformation, which is carried out

in the more efficient and cheaper way. Prakash and his colleagues (2011) generated a

unique device, which is a 30 centimeters long explosive coated polymer tube to create a

micro-shock wave that facilitate the entry of DNA from the surrounding into the cells of

bacteria. Prakash et al. (2011) believed that the momentum disturbance that resulted from

the shock wave can change the bacterial cell membrane permeability to facilitate the

uptake of the DNA from the surrounding. According to Parkash et al. (2011), the

efficiency of transformation by using this method is higher as electroporation technique,

13

and it has a benefit of better recovery of cells, and it is rather cheaper than the commercial

electroporation.

Figure 5: Schematic diagram of the micro-shock wave devices.

(Adapted from: Prakash et al., 2011).