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RECONSTRUCTION OF msAdh GENE

Ho Carl Miew

A project submitted in partial fulfillment of the requirement for the degree of Bachelor of Science with Honours

(Resource Biotechnology)

Department of Molecular Biology Faculty of Resource Science and Technology

Universiti Malaysia Sarawak 2011

Acknowledgement

111

I would like to grab this opportunity to express my sincere appreciation and greatest

gratitude to my supervisor, Associate Professor Dr. Hairul Roslan for allowing me to work

on the Final Year Project entitled Analysis of Adh protein expression in bacterial system

and for giving me his invaluable and consistent guidance advice, and supports throughout

the project. I am grateful to be part of the members of Genetic Engineering Lab.

I would like to extend appreciation to my co-supervIsor, Dr. Azaham, for brilliant

suggestion and advice. My gratitude also goes to lecturers of faculty of Resource Science

and Technology especially Associate Professor Dr. Hasnain, Associate Professor Dr.

Awang and Dr. Lee for their kindness supports and advice.

My heartiest gratitude to all the postgraduates, especially to Jerry Gerunsin, N abella

Holling, Liyana Ismail and Nickson from Proteomics Lab for their companionship,

patience guidance, kindness encouragement and wonderful experience we shared in the lab.

Last but not least, I am deeply appreciated my family and friends for their never ending

supports and love.

I

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Acknowledgement

Declaration

Table of content

List of Abbreviations

List of Tables

List of Figures

Abstracts

1.0 Introduction

2.0 Literature Review

3.0 Material and

Methods

Table of Content

I

II

III

VI

VII

VIII

1

1.1 Background 2

1.2 Objective 5

6

2.1 Alcohol dehydrogenases (Adh) genes 6

2.2 Cloning and Expression Hosts 8

2.3 Gene Cloning 10

2.4 Blunt-end Ligation Reaction 11

13

3.1 Preparation of Luria-Bertani (LB) 13

3.2 Amplification and Extraction of Plasmid 13

3.3 Verification ofAdh gene via Polymerase Chain 14

Reaction (PCR) and Restriction Enzyme (RE)

Analysis

3.4 Blunt-end Reactions 15

III

4.0 Results

,.

3.5 Ligation Reactions 16

3.6 Verification of Blunt-end Ligation reaction via Two 16

Steps PCR using Different Specific Primers

3.7 Overnight E. coli cell Culture Preparation and 17

Preparation of Competent Cells via Heat Shock

Method

3.8 Transformation ofpET41a (+) with Incorporated Adh 17

Gene into E.coli Bacterial System

3.9 Directional Cloning of PCR products 18

3.1 0 Verification of Clones via Colony PCR 19

3.11 Cultivation ofClones and verification via PCR 19

3.12 Sequencing ofAdh/pET gene sample 20

21




Analysis

4.3 Restriction Enzyme (RE) Digestion using NdeI and 24

BgnI Endonuclease for Blunt-end Ligation Reaction

4.4 Verification of Blunt-end Ligation reaction via 26

Transformation into E.coU Bacterial System

4.5 Verification of Blunt-end Ligation reaction via PCR 27

using Different Specific Primers II 4.6 Polymerase Chain Reaction (PCR) using 5'NdeI- 27

adaoptor and 3'XhoI-adaptor Specific Primers

followed by Restriction Enzyme (RE) Digestion via

NdeI and XhoI Endonuclease and Ligation Reaction

IV

p fP

5.0 Discussion

6.0 Conclusion

7.0 References

8.0 Appendices

•

4.7 Verification of Clone via Colony PCR 28

294.8 Cultivation ofClones and Verification via PCR

304.9 DNA Sequencing Analysis ofA dh/pET gene

33




Analysis

5.3 Restriction Enzyme (RE) Digestion using NdeI and 35

Bgm Endonucleases for Blunt- end Ligation

Reactions

5.4 Verification of Blunt-end Ligation Reaction via PCR 36

using Specific Primers

5.5 Polymerase Chain Reaction (PCR) using 5'NdeI- 37

adaptor and 3'XhoI-adaptor Specific pdmers

followed by Restriction Enzyme (RE) Digestion via

NdeI and XhoI Endonuclease and Ligation Reaction

5.6 Verification of clone via Colony PCR 38

5.7 Cultivation ofClones and Verification via PCR 38

5.8 Analysis of DNA Sequencing ofAdhipET Gene 39

42

43

49 I

v

11

I

List of Abbreviations

°C Degree Celsius

d Dalton

g Gram

M Molar

mM MiliMolar

ml Mililiter

mg/ml Miligram per mi11iliter

ilL Microliter

Ilg/L Microgram per Liter

rpm Round per Minute

EDTA Ethylenediaminetetraacetic acid

CaCh Calcium Chloride

NaCl Sodium Chloride

KCI Potassium Chloride

DTT Dithiothreitol

PBS Phosphate buffered saline

VI

List of Tables

Table Page

1. BLASTn output for AdbJpET sequence of approximately 1000 bp. 39

2. BLASTn output for AdbJpET foreard sequence of approximately 800 bp. 41

3 Various parameters of blunt-end ligation reactions 49

4. pET41 (+) restriction sites that involved in this study 51

I

VII

7

List of Figures

....-,. ,

Figure Page

1. Alcoholic fermentation

2. The picture shows the results of verifying plasmid extraction via 0.8% agarose 21

gel.

3. The picture shows the results of single digestion usmg XbaI (10 U/)lL) 22

endonuclease at 0.8% agarose gel.

4. The picture shows the results of PCR of Adh/pET using Adhmor8-f and 23

Adhmor8-r specific primers at 0.8% agarose gel.

5. The picture presents the result ofRE double digestion using NdeI (10 U/)lL) and 23

BgnI (10 U/)lL) endonuclease at 0.8% agarose gel.

6. The picture shows the results of restriction enzyme double digestion using NdeI 24

(10 U/)lL) and BgnI (10 U/)lL) endonuclease for first attempt of Blunt-end

Ligation reaction.

7. The picture indicates the results of double digestion using NdeI (10 U/)lL) and 25

BgnI (10 U/)lL) endonuclease for the second attempt of blunt-end ligation

reaction.

8. The picture shows the verification of double digestion of NdeI (10 U/)lL) and 25

BgnI (10 U/)lL) endonuclease for third attempt of Blunt-end Ligation reaction

by running on 0.8% agarose gel.

9. The picture shows the result of double digestion using NdeI (10 U/)lL) and BgnI 26

(10 U/)lL) endonucleases for fourth attempt of Blunt-end Ligation reaction.

10. The picture shows the results of PCR products using 5'NdeI-adaptor and 28

3 'Xhol-adaptor specific primers and restriction enzyme analysis using NdeI and

VIII

I

A'hoI endonucleases.

11. The picture shows the results of PCR using different sets of primers. The 29

verification was carried out by running on O.S% agarose gel.

12. The picture shows the results of PCR products using AdhmorS-f and AdhmorS-r 30

specific primers. The verification was carried out by running on O.S% agarose

gel.

13. The picture shows forward sequence with the perfect gap juntion of NdeI at 51 31

bp was viwed via ChromasPro software.

14. The picture shows the reverse-complemented sequence with the correct gap 31

juntion ofA'hoI at 942 bp.

15. The picture shows the contig sequence generated by overlapping forward and 32

reverse-complemented reverse sequence via CAP3 online program.

16. The figure shows the overlapping sequence of forward and reverse sequencing 32

results using CAP3 online program. The overlapping region composed of

approximately 300 bp.

17. Partial part of the chromatogram of Adh/pET forward sequence with significant 40

result.

IS. The picture shows the BLASTn outcome of Adh/pET forward sequence which 40

consists of approximately 300 bp.

19. pET 4la (+), Novagen with the cloning sites. 50

20. pET 41 a (+) vector's cloning or expression regions. 51

IX

ABSTRACT

Alcohol dehydrogenase (Adh) genes are highly characterized in alcoholic pathway which normally occurs in most of plants. These genes are activated when under anaerobic stresses by producing measurable level of alcohol dehydrogenase enzyme which is capable in converting acetaldehyde into relatively low toxicity compound, ethanol. This vital response of plants has been proved in enhancing the potential for survival under hypoxic conditions with a low production of A TP and regeneration of NAD+· The objective of this study was to identify and verify the presence of AdhipET inserts within plasmid which is isolated from Metroxylon sagu via Restriction endonuclease and Polymerase Chain Reaction. In addition, reconstruction of the msAdh plasmid was carried out due to the frame shift of single nucleotide.

Key words: Alcohol dehydrogenase (Adh) genes, Metroxylon sagu, coli BL21 (DE3)

ABSTRAK

Gen alkohol dehydrogenase sering dikaitkan dengan proses fermentasi alkohol yang biasa berlaku pada tumbuh-tumbuhan. Gen-gen terse but diaktifkan semasa dalam stress anaerobik dengan menghasilkan enzim alkohol dehydrogenase pada tahap yang boleh dikesan yang berfungsi mensintesiskan etanol yang kurang bertosik daripada acetaldehyde. Respon tersebut telah dibuktikan berupaya meningkatkan potensi untuk kelangsungan hidup semasa menghadapi situasi hipoksia dengan bekalan A TP yang rendah serta regenarasi NAD+. Objektif kajian ini adalah untuk mengenalpasti dan mengesan kehadiran gen AdhipET dalam plasmid yang diasing daripada Metroxylon sagu melalui cara-cara seperti anal isis enzim pembatas and reaksi polimeras berantai. Tambahan pula, rekonstruksi plasmid msAdh dilaksanakan oleh kerana berlakunya frame shift oleh kehadiran nukleotida tunggal.

Kata kunci : Gen alkohol dehydrogenase, Metroxylon sagu, E. coli BL21 (DE3)

1

1.0 Introduction

1.1 Background

Metroxylon sagu or sago plam is commonly known as rumbia that is commercially

grown in Malaysia (McClatchey et ai., 2006). McClatchey and colleagues (2006) also

claimed that Metroxylon species especially Metroxylon sagu, is suitable to be grown in

tropical rainforest of South-East Asia and the equatorial Pacific. Typically, sago palm

plantations are distributed in areas that are inapt for agriculture activity, yet, sago palm

cultivation is often the most suitable and ecologically appropriate form of the unsuitable

land condition (Sundaraj, 2008). In Malaysia especially the state of Sarawak, sago palm

plantations are cultivated in large areas whereby not only considering its economical

aspects, but also an appropriate form for land-use (Sundaraj, 2008). The low tolerance of

water shortage for Metroxylon sagu requires ample and uniform rainfalls throughout the

years (McClatchey et al., 2006). Metroxylon sagu is hapaxanthic which means it only

flowers once during its life; yet Metroxylon sagu is under the plant category of easy

vegetative multiplication (Orwa et al., 2009). Sago palm has been extensively studied as to

convert sago starch into alternative products such as fuel ethanol. Apart from that, sago

palm can also be used as food supplement, fiber, and other beneficial products in the

market (Orwa et ai., 2009). Production of ethanol by most of the plant including

Metroxylon sagu under anaerobic condition is followed by alcoholic fermentation pathway

(Strommer & Garabagi, 2009).

Oxygen availability is one of the curial limiting factors for plant growth and

survival especially in flooded soils (Harry & Kimmerer, 1990; Subbaiah & Sachs, 2009). A

serious flooding incident happened in 1993 which led to serious economic loses especially

the large areas of corn and soybean plantations in Midwestern United States (Subbaiah &

Sachs, 2009). Tremendous attentions and efforts are being contributed to understand the

2

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responses of crops to predominant stress such as oxygen deprivation and improve flooding

tolerance of the crops (Harry & Kimmerer, 1990; Subbaiah & Sachs, 2009; Arru &

F omaciari, 20 I 0). When oxygen becomes the limiting factor of either in growth or survival

of flooded plants, both the morphological and physiological adaptations of plants are

dramatically affected (Sundaraj, 2008; Subbaiah & Sachs, 2009). Genetics studies have

shown evidence of anaerobic induction of maize Adh I leads to elevation of transcripts

stability, thus, changing the chromatin structure (Harry & Kimmerer, 1990). Nevertheless,

the predominant anaerobic stresses that flooded plants are include anoxia and hypoxia.

Harry and Kimmerer (1990) defined anoxia as condition occurs when there is absolute

absence of oxygen, whereby truly anoxic condition rarely happen in nature and only few

plants can survive under such condition. In contrast, hypoxia occurs when there is

insufficient or partial depletion of oxygen supplied (Harry & Kimmerer, 1990; Subbaiah &

Sachs, 2009).

Various studies have been done in demonstrating the evolution of different

surviving methods of plants under anaerobic stresses. When plants are under oxygen

tension, they overcome the flooding stresses by: (1) ethanol fermentation compensating the

reduced energy yield, (2) formation of relatively low toxicity or non-toxic end products, (3)

transportation of oxygen from atmosphere to roots (Rozema & Verkleij, 1991; Li et al.,

2001; Sundaraj, 2008). In addition, Subbaiah and Sachs (2009) claimed that plants also

suffer from insufficient oxygen supply under normal development which in tum affects the

reproductive development of the plants. According to Subbaiah and Sachs (2009), this is

due to the immense biomass and it's greatly reliance on diffusion through intercellular

spaces for oxygen supply of the plants when under non-flooding conditions. Under the

condition of insufficient oxygen, oxidative phosphorylation is inhibited and the amount of

adenosine triphosphate (A TP) production is low. Therefore, pyruvate decarboxylase (PDe)

3

"" ,....

and alcohol dehydrogenase enzymes (AD H) via the ethanolic fermentation produce ethanol

as end-product for anaerobic glycolysis. In addition, a low level of energy production in

the form of A TP can be maintained with also regenerating small amount of nicotinamide

adenine dinucleotide (NAD+). The ethanolic fermentation pathway is an alternative to

enhance the survival of the plant when it encounters any hypoxic stress or disruption of

mitochondrial activity (Strommer & Garabagi, 2009).

Nevertheless, various studies have been focused on Adh genes activities to fit the

pattern of anaerobic stress response. The end product of Adh genes is alcohol

dehydrogenase (ADH) enzyme, which is an anaerobic enzyme that converts acetaldehyde

into ethanol and ATP furthermore preventing the plant from acidosis (Strommer &

Garabagi, 2009). The conversion pathway of acetaldehyde into ethanol with the presence

of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) enzyme results in

continuous NAD+ regeneration (Sundaraj, 2008; Strommer & Garabagi, 2009). Alcohol

dehydrogenase (ADH) is a dimeric-zinc enzyme, which is also categorized as isozyme in

most of the plants (Tesniere & Abbal, 2009). Most of the plants carry more than one Adh

genes which may have different expressions of protein in various organs at particular times

either during period of developmental or responses to environmental condition (Harry &

Kimmerer, 1990). Tensniere and Abbal (2009) stated that the evolution of the enzyme's

activity as well as gene expression in plant kingdom for various plant organs have been

widely studied in response to environmental stress, such as anaerobiosis and hypoxic.

Apart from being considered as stress-response markers, Adh gene also shows others

capabilities such as fruit-ripening related markers (Tesniere & Verries, 2000).

In order to investigate Adh gene, Escherichia coli is selected as the bacterial system

in this study. Escherichia coli is commonly referred as E. coli. It is a gram negative,

facultative anaerobe which can generate energy in the form of A TP via respiration and

4

I

fermentation (Goodlove e/ al., 1989). E. coli is the first host used to express recombinant

protein and is one of the most characterized bacterial systems in expressing foreign protein.

High frequency in using E. coli system in expressing recombinant DNA is due to several

advantages: E. coli system offers rapid and high-level expression with short doubling time,

yet, the growth media are inexpensive and low complexity (Cantrell, 2003; Brondyk, 2009).

Apart from that. E. coli system is able to target protein in desired location (Cantrell, 2003;

Brondyk, 2009).

1.2 Objectives

Proteins are one of the vital molecules mostly involved in biological processes. In

term of economical and environmental factors, low-cost fuel ethanol production is

necessity in order to sustain the development of country (Rogers e/ aI., 2007; Jeon et ai.,

2008). Studies are carried out in order to understand the fundamental functions of Adh

genes involved in anaerobic mechanism pathway. The objective of this study was to

reconstruct the msAdh. The Adh gene which ligated in pET41a (+) vector in the previous

study, which is msAdh was in an incorrect frame. Therefore, reconstruction of the gene was

carried out via E. coli XLI Blue. The verification of the msAdh reconstruction was done by

plasmid DNA sequencing.

5

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2.0 Literature Review

2.1 Alcohol Dehydrogenase (Adh) gene

Alcohol dehydrogenases (Adh) are active as dimeric-zinc enzymes which encode

for glycolytic protein have been extensively studied in various organism (Thompson el ai.,

2007; Tesniere & Abbal, 2009). Generally, ADH is known for its function by reducing

toxic acetaldehyde into ethanol. In order to survive under hypoxic stress, pyruvate

dehydrogenase enzyme (PDC) is responsible in catalyzing the initial anaerobic

fermentation by converting pyruvate, which is generated from glycolysis to acetaldehyde

and further reduced to ethanol by Adh (Talarico el ai., 2005; Strommer & Garabagi, 2009).

According to Strommer and Garabagi (2009), PDC enzyme explicates a critical role

when under anaerobic condition by carrying out the first step of non-oxidative

decarboxylation. During the initial stage of non-oxidative decarboxylation acetaldehyde is

supplied to ADH enzyme, producing enough A TPs to protect flooded plants from acidosis

(Talarico et aI., 2005; Strommer & Garabagi, 2009). However, ethanol production in most

plants in favor to other substances such as, lactate is mainly due to several advantages of

production of ethanol as end product of anaerobic metabolism (Harry & Kimmerer, 1991).

The benefits include ethanol exhibits relatively low-toxicity hence ease of elimination from

the plant cells (Harry & Kimmere, 1991). Most importantly, ethanol also plays a critical

role in stabilizing intracellular pH (Harry & Kimmerer, 1991). Figure 1 shows the coupling

system of PDC and ADH enzymes when the oxidation phosphorylation is inhibited due to

hypoxic stress.

6

PDH

Glucose P'11Jyute ----~Acetyl-CoA -~Krebs cycle

I 1

PlJC ~

Acetnldeh,'de .·tIIll :,,. Ethanol

(Deficient in O:\.'ygen)

Figure 1, Alcoholic Fennentation (Strommer & Garabagi, 2009)

The amino acid sequences of Adh genes are highly conserved, however, due to the

senes of duplications and functional divergences events, the Adh variants and their

metabolic functions are variable (Yokoyama et aI., 1990; Dolferus et aI., 1997; Thompson

et al., 2007; Strommer & Garabagi, 2009; Tesniere & Abbal, 2009). Based on phylogenetic

analysis, various studies proved that Adh genes have had undergo evolution events

subsequently replacement of adaptative amino acid of Adh genes (Batterham et aI., 1983;

Thompson et al., 2007). These genes are labeled as Adhl, Adh2 and Adh3 which are either

homologous or heterologous (Thompson et al., 2007). Research carried out by Pal and

colleagues (2009) on Saccharomyces carlsbergensis showed that, yeast contains Adh1 and

Adh2 and both Adhl and Adh2 are in cytoplasmic form. However, Adhl is proved to

deplete the acetaldehyde compounds in glucose fermentation; whereas, Adh2 is responsible

for oxidizing ethanol in aerobic metabolism (Jomvall, 1977; Bennetzen & Hall, 1982;

Young et aI., 1983; Pal et aI., 2009). As for Adh3 gene in yeast, it is in mitochondrial form

which involves in reducing equivalents ofNADH (Ganzhorn et aI., 1987; Pal et al., 2009).

The first studied of Adh genes activities in plant of Berger and Avery (1943) is on

Adh activities in extracts of oat coleoptiles (Strommer & Garabagi, 2009). Subsequently,

researches carried out studies on different parts of plant kingdom by demonstrating Adh

7

genes activities under various stress or anaerobic conditions, such as dehydration,

wounding and low temperature (Kimmerer & Kozlowski, 1982; Nogchi, 2001; Seki et al.,

2002; Strommer & Garabagi, 2009). Adh genes activities are detected at different parts of

the plant, yet, demonstrating diverse responses on anaerobic conditions. Petunia hybrida is

the case in point, which shows Adh2 genes activities in both styles and the ovaries respond

differently (Strommer & Garabagi, 2009). As for Adh2 gene activities in styles, it functions

as the agent of pollen tubes development, which requires induction of pollination

(Garabagi & Strommer, 2004; Garabagi & Strommer, 2005). However, Adh2 genes

expression is likely to be associated with the hypoxia in ovaries (Linskens & Schrauwen,

1966; Strommer & Garabagi, 2009).

2.2 Cloning and Expression Host cells

E. coli is the popular choice especially for heterologous gene expression cloning

works (Kunze et al., 1995; Freuler et al., 2008). The widespread use of E. coli system in

expressing recombinant protein in both laboratory and industrial process scale is not only

due to nominal cost yet high-level expression, but also the capability in enhancing the

recombinant protein expression especially cytoplasmic expression (Baneyx, 1999; Cantrell,

2003; Terpe, 2006). This is due to the benefits of E. coli carry which is useful for

expressing large distinct domains that is up few thousand amino acids (Freuler et al., 2008).

In addition, the codon usage and regulatory sequences of E. coli seldom interrupt the

heterologous gene expression (Freuler et al., 2008). However, the strain or genetic

background for recombinant expression is important as to maintain stability of plasmid

expression and bacterial system expression (Sorensen & Mortensen, 2004). E. coli BL21

strain was first developed by Studier and Moffatt in t986, which is commonly practiced in

studying the T7-dependent expression of recombinant protein (Cognet et ai, 2003).

Sorensen and Mortensen's research in 2004 indicated that E. coli BL21 strain is proven to

8

have outstanding recombinant expression applications. BL21 is a robust E. coli B strain, a

lysogen of DE3 bacteriophage that contains T7 polymerase gene, which enable vector such

as pET plasmid family to produce recombinant protein (Studier et al., 1990; Cognet et al.,

2003). Moreover, another advantage of using E. coli BL21 strain is that the culture can be

grown in minimal media, which requires low cost preparation (Chart et al., 2000).

Nevertheless, there are several drawbacks of using E coli as cloning host cells as

such lacking of eukaryote-specific protein modification subsequently results in incorrect

folding of heterologous protein and subsequently accumulation of insoluble proteins

(Kunze et al., 1995). Nonetheless, expression of proteins especially for toxic protein,

plasmid instability may arise. The cloning steps for integrating the region of interested into

vector should be performed in E. coli strain lacking T7 RNA polymerase gene, such as

XLI Blue coli (Kunze et aI., 1995; Mierendorf et al., 2000). This is to eliminate plasmid

instability that due to the production of proteins, which is potentially toxic to host cells

(Kunze et aI., 1995; Mierendorf et al., 2000). Moreover, verification of desired inserts

present within the plasmid need to be carried out by transforming it inside E. coli, followed

by other verifying procedures. Only the final construct is transformed into the expressing

host, for instance pLysS and pLysE host cells (Kunze et al., 1995). The stability of plasmid

in expressing target gene in pLysS and pLysE host cells is achieved due to the presence of

T7 lysozyme gene and T7 promoter gene (pET Manual System 8th Edition, 1999; pET

Manual System loth Edition, 2003;).

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2.3 Gene Cloning

Gene cloning has played an important role especially in science industries. Gene

cloning is a highly characterized technique in identifying a pure sample of an individual

gene (Brown, 1995). Coupling with gene cloning, techniques such as transformation and

preparation of competent cells are also the keys to the success of a cloning experiment

(Brown, 1995). Nevertheless, other aspects including choosing a suitable vector and host

cells are also critical steps. Generally, vectors can be divided into two categories which are

8thtranscription vector and translation vector (PET Manual System Edition, 1999).

Eukaryotic genes are usually cloned in translation vectors (Zerbs et af., 2009). This is

because the absence of the ribosome-binding site results an incompatibility with E. coli

translation machinery (pET Manual System 8th Edition, 1999; Zerbs et af., 2009). In

addition, translation vectors that derived from pET system are usually used for the

expression of target genes without the ribosomal binding site (PET Manual System 8th

Edition, 1999; pET Manual System 10th Edition, 2003; Zerbs et ai., 2009). Different from

translation vectors, transcription vectors are normally for target gene that already consists

of prokaryotic ribosome binding site and A TG start codon (pET Manual System 8th Edition,

1999). However, there are three considerations need to be taken account of when choosing

the suitable pET vector including the application intended for the expressed protein,

cloning strategy and specific information of the expressed protein.

One of the most used engineered vectors to expressing recombinant protein is the

T7 based pET vector series (Soresen & Mortensen, 2004; Zerbs et af., 2009). Sorensen and

colleagues (2004) stated that a strong transcriptional promoter is vital for recombinant

protein expression to obtain high-level protein expression. The commercial pET vector

series (Novagen), pET 41 a (+) with the presence of T7 promoter, is the chosen vector for

Adh expression. E. coli strain which has the ability to express T7 RNA polymerase or also

10

known,as T7 DNA-dependent RNA polymerase (T7 RNAP) is essential for recombinant

protein expression when pET vector series is used (Durbin, 1999 ; Zerbs et al., 2009).

Directional cloning of PCR product was also demonstrated in this study by using a

pair of specific primers, Adhmor8-f and Adhmor8-r. Generally, this method is

accomplished by integration of restriction sites in 5' end of primers, hence followed by

restriction enzyme digestion of the products (Gal & Kalman, 2002; Xie & Xie, 2011).

Sticky-ends fragments are generated as the end product and further ligated into compatible

vectors' overhangs (Gal & Kalman, 2002; Xie & Xie, 2011). Nevertheless, type II

restriction endonucleases cleave rather inefficiently of the cleavage sites which are too

close to DNA termini (Gal & Kalman, 2002; Xie & Xie, 2011). These are enhanced by

adding nucleotides which is irrelevant to the sequence or the desired inserts to the 5' end of

each primer (Xie & Xie, 2011). Another Apart from that, due to the presence of internal

restriction sites of the target fragments may complicate the cloning work (Gal & Kalman,

2002; Xie & Xie, 2011).

2.4 Blunt-end Ligation Reaction

Construction of chimeric DNA molecules is one of the major techniques in

recombinant technology, which greatly depends on the efficiency and ability of restriction

endonuclease. Most of the restriction endonucleases function as cutter for interest

restriction sites by catalyzing breakage of the phosphodiester linkage for further joining

target fragments (Song et al., 1985). Nevertheless, T4 DNA Polymerase and Klenow

Fragments have the ability in producing blunt-end reaction. The subsequent step is the

most important criteria in generating blunt-end ligation via T4 Ligase (Song et ai., 1985).

The efficiency of ligation reaction especially blunt-end ligation reaction depends on several

parameters including temperature and period of incubation, ionic concentration, presence

11

of excessive salt and A TP amount, efficiency and amount of ligation enzyme (Sugino et al.,

1977; Deugau & Sande, 1978; Song et al., 1985). According to Song (1985), Sugino and

colleagues (1977), the efficiency of blunt-end ligation reaction is greatly enhanced by

prolonging incubation period and with the presence of PEG 6000. They also claimed that 1:

3 or 1: 5 molar ratios of inserts-vectors ligation are most recommended.

Sharp et af. (1970) and Song et af. (1985) reported that T4 DNA Ligase is also

widely demonstrated not only in the generating closure of nicks, but also joining the

double-stranded DNA at base paired ends. Sharp et af. (1970) and Song et al. (1985) also

suggested that the ligation reaction is sharply improved by extending incubation period at

maximal 4°C. They reported that incubation temperature of ligation with higher than 4°C

interferes with the ligation reaction to work at optimal efficiency. They claimed that this is

due to the presence of contaminants. In addition, increased concentration of A TP and

monovalent cation such as Na+ inhibit the ligation (Sugino et al., 1977; Deugau & Sande,

1978; Song et aI., 1985). Heat inactivation ofT4 Ligase is critical before proceeding to the

transformation of recombinant molecules into host cells is claimed can elevate the

transformation efficiency (Song et af., 1985; Smith, 1993; Fermentas, 2011).

12

T 3.0 Materials and Methods

3.1 Preparation of Luria-Bertani (LB)

Total of 50 mg/ml of kanamycin was prepared by adding 109 kanamycin into 10

ml of deionised water. The working stock of kanamycin was aliquot to 1 ml and store at

20°C until it was used. Luria-Bertani media (2 g Tryptone; 1 g yeast extract; 1 g NaCI; 200

ml deionised water) with the presence of kanamycin was prepared in the volume of 2x500

ml for two samples. Kanamycin (1 ml of 50 mg/ml) was added to make 10 mg/ml Luria

Bertanil kanamycin. The Luria-Bertani was added with extra 15 g agar and kanamycin was

then added to the Luria-Bertani media when it was cooled. Then, 20 ml of the mixture was

poured into the plastic petri dishes (Sam brook et al., 1989).

3.2 Amplification and Extraction of Plasmid

Escherichia coli XL I Blue with the presence of the insert, AdhipET were cultured

for 22 hours at 3JOC in Luria-Bertani media (Sambrook et al., 1989). The overnight

plasmid culture was cultured for another two hours in 1 ml of fresh Luria-Bertani with 1

mg/ml of kanamycin antibiotic furthermore verified via runnig the Agarose Gel

Electrophoresis at 0.8% of gel concentration. Later, the extracted plasmid from the

remaining overnight culture was also ran on Agarose Gel Eelectrophoresis at 0.8%

concentration. Both of the expected band sizes are seven kb (Sam brook et al., 1989;

Vivantis, 2009; Smith, 1993).

13

T 3.3 Verification of Adh gene via Polymerase Chain Reaction (PCR) and Restriction

Enzyme (RE) Analysis

The extracted plasmid was verified by running PCR with three set of primers,

included (1) Adhmor8-f (5'-CTAGAGCTTCAGGGGCATCA-3') and Adhmor8-r (5'

ACGCAAGGGA AGGCTAAGAT-3') specific primers, which ran for thirty cycles at

95°C for 15 sec, 65°C for 15 sec and noc for 30 sec, (2) 5'NdeJ-adaptor (5'

GGAATTTATGGCAAGCAG TGTTGG-3') and 3'A'hoI-adaptor (5'

CCGCTCGAGTTTTTT TTTTTTTTTTTT-3') primers, which was ran for thirty cycles at

95°C for 15 sec, 59°C for 45 sec and noc for 1 minute and 30 sec. and (3) T7 promoter 59

(5'TTAATACGACTCACTATAGGGG-3') and T7 terminator 59 (5'ATGC TA

GTTATTGCTCAGGGGT-3') that ran for thirty five cycles at 95°C for 15 sec, 59°C for 45

sec and noc for 1 minute. Master mix of total volume of 20 ~L was prepared, included 10

~L of GoTaq Green master mix (2X), 7 ~L of ddH20, 1 ~L of each primer and 1 ~L of

DNA template. Together with the preparation of negative control, total of two samples

were prepared to run the PCR. The PCR products were then ran on the Agarose Gel

Electrophoresis at 0.8% of gel concentration. The expected band size is seven kb

(Sambrook et ai., 1989; Smith, 1993).

Verification was also carried out via double restriction enzyme digestion. Total of

20 ~L of mixture reaction was prepared with 15 ~L of DNA template, 2 ~L of Orange

buffer (lOX), I ~L of each of NdeI (1 OUl~I) and BglIl (1 OUl~l) restriction enzymes and 1

~L of ddH20. The reaction mixture was then incubated at 37°C for three hours. After three

hours incubation, the restriction enzyme reaction was verified via Agarose Gel

Electrophoresis at 0.8% of gel concentration. The expected bands sizes were 5.5 kb and 1.3

kb. The DNA from the double restriction enzyme digestion was recovered by using the

GF-I DNA Recovery kits from Vivantis. Single digestion using XbaJ (1 OU/~I) was also

14

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performed to further verify the insert in the plasmid and further ran on Agarose Gel

Electrophoresis at 0.7% of gel concentration. The expected band size is eight kb

(Sambrook et ai., 1989; Smith, 1993).

3.4 Blunt-end Reactions

The DNA templates recovered from agarose gel were subsequently used in the

blunt-end reaction with the total reaction volume of 20 IiL volume that included 15 IiL of

DNA template, 2 IiL of T4 Polymerase Buffer (lOX), I IiL of dNTPs (2mM), 1 IiL of T4

DNA Polymerase (5U/IiL). Before ligation reaction was carried out, the blunt-end reaction

mixture was incubated at 11°C for twenty minutes, followed by heat inactivation at 75°C

for ten minutes.

A second attempt of blunt-end reaction was performed with modification on the

protocol by increasing amount of T4 DNA polymerase up to 2 IiL (5U/IiL) and allowed to

incubate at 11°C for sixty minutes. Later, third attempt was performed using new T4 DNA

polymerase. Total of 2 IiL of T4 DNA polymerase (5U/IiL) was added to the blunt-end

reaction mixture, and was incubated at 11°C for sixty minutes. Both second and third

attempt were underwent heat inactivation at 75°C for ten minutes before carrying out

ligation reaction. Last attempt of blunt-end reaction was performed which was similar with

the third attempt of blunt-end reaction. However, there was a slight modification in ligation

reaction which is further discussed in section 3.5 ligation reactions (See page 16) (Song et

aI., 1985; Sambrook et al., 1989; Fermentas, 2011).

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