CLONING OF Candida antarctica LIPASE A GENE IN Kluveromyces lactis EXPRESSION SYSTEM

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CLONING OF Candida antarctica LIPASE A GENE IN

Kluveromyces lactis EXPRESSION SYSTEM

A PROJECT REPORT

Submitted by

KARTHIKEYAN.T

RAJARAJAN.P

RAMKUMAR.M

In partial fulfillment for the award of the degree

of

BACHELOR OF TECHNOLOGY

in

INDUSTRIAL BIOTECHNOLOGY

CENTRE FOR BIOTECHNOLOGY

ANNA UNIVERSITY

CHENNAI – 600025

DECEMBER 2009 – MAY 2010

ANNA UNIVERSITY: CHENNAI 600 025

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BONAFIDE CERTIFICATE

Certified that this project report “Cloning of Candida antarctica Lipase A gene in

Kluveromyces lactis expression system” is the bonafide work of Karthikeyan.T

(Reg.No.20062735), Rajarajan.P (Reg.No.20062760) and Ramkumar.M

(Reg.No.20062761) who carried out the project work under my supervision.

SIGNATURE SIGNATURE

Dr.P.Kaliraj Dr.S.Meenakshi Sundaram

Professor and Head Professor,

Centre for Biotechnology, Centre for Biotechnology,

Anna University, Anna University,

Chennai-25. Chennai- 25.

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ACKNOWLEDGEMENT

We sincerely thank our project guide Dr.S.Meenakshisundaram, Professor,

Centre for Biotechnology, Anna University for allowing us to do our project in the

field of molecular biology in bioprocess. He gave us full freedom to try new

techniques and to conduct our own experiments.

We would like to thank Ms.A.K.Prasanna Vadhana for assisting us

throughout the project. We really appreciate her effort in trying to help us in our

project.

We are happy to get acquainted with other research scholars in bioprocess

lab. They were extremely helpful and enabled to create a comfortable working

atmosphere.

We also extend our thanks to other fellow students whose guidance and

views have gone in to the completion of this project.

Karthickeyan T Rajarajan P Ramkumar M

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TABLE OF CONTENTS

CHAPTER NO. TITLE PAGE NO.

ABSTRACT 6

LIST OF FIGURES 7

LIST OF TABLES 8

LIST OF SYMBOLS 8

1 INTRODUCTION 10

1.1 LIPASES 10

1.2 BIOCHEMISTRY OF LIPASES 10

1.3 LIPASE ACTIVITY ASSAY 12

1.4 REVIEW OF LITERATURE 13

1.4.1 Candida antarctica LIPASES 13

1.4.2 INDUSTRIAL APPLICATIONS 14

OF LIPASES

1.5 E.coli MAINTANENCE HOST 16

1.6 K.lactis EXPRESSION SYSTEM 16

2 MATERIALS AND METHODS 19

2.1 HOST STRAINS 19

2.2 VECTORS 19

2.3 ANTIBIOTICS 19

2.4 CULTURE MEDIUM 20

2.5 GENE OF INTEREST 20

2.6 POLYMERASE CHAIN REACTION 20

2.7 AGAROSE GEL ELECTROPHORESIS 22

2.8 RESTRICTION 23

2.9 PURIFICATION 25

2.10 STOCK PREPARATION 26

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2.11 INOCULATION 26

2.12 PLASMID EXTRACTION 26

2.13 RESTRICTION 29

2.14 DEPHOSPHORYLATION 30

2.15 PURIFICATION 31

2.16 LIGATION 32

2.17 COMPETENT CELLS PREPARATION 36

AND TRANSFORMATION

2.18 TRANSFORMATION 38

2.19 PATCHING 40

2.20 LYSATE PCR 40

2.21 CONFIRMATION PCR 41

3 RESULTS AND DISCUSSION 43

3.1 PCR AMPLIFICATION 43

OF C.ANTARTICA LIPASE A

ENCODING GENE

3.2 RESTRICTION AND PURIFICATION 44

OF LIPASE A GENE

3.3 pKLAC2 – EXTACTION AND RESTRICTION 45

3.4 CLONING 47

3.4 TRANSFORMATION 48

4 CONCLUSION 51

REFERENCES 52

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ABSTRACT

Lipase A is an enzyme from Candida antarctica strain isolated from Lake

vanda in Antarctica. The gene coding for Lipase A is CALA gene. Lipase A is the

most thermostable lipase known and is used as a biocatalyst in food and

pharmaceutical industry. Lipases (triacylglycerol acylhydrolases EC 3.1.1.3) are

ubiquitous enzymes of considerable physiological significance and industrial

potential. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and free

fatty acids. Lipases are serine hydrolases. Lipases display little activity in aqueous

solutions containing soluble substrates.

Lipase A gene was from Candida antarctica was amplified using gene

specific primers Xho1 restriction site. The gene was cloned unidirectionally into

Xho1 site in pKLAC2 vector. pKLAC2 with lipase A gene insert was transformed

into E.Coli TOP 10F’ strain and the transformed colonies were screened for

positive transformants. Future prospects involve expressing lipase A gene in

Kluveromyces lactis expression system and conducting expression studies in a

fermenter.

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LIST OF FIGURES

CHAPTER NO TITLE PAGE NO

1 1.1 Mechanism of lipase catalysis 11

1.2 pKLAC Vector Map 18

2 2.1 Plasmid Extraction 29

2.2 A pictorial example of how a 33

ligase works (with sticky ends)

2.3 A simple mechanism 34

representing the role of DNA Ligase

2.4 Artificial Transformation 39

3 3.1.1 Analysis of PCR amplified 44

lipase A DNA of Candida antarctica

3.2.1 Analysis of purified sample 45

of Lipase A gene

3.3.1 Analysis of PKLAC2 plasmid 46

extracted by standard kit method

3.3.2 Analysis of pKLAC2 restricted Plasmid 47

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3.4.1 Analysis of ligation mixtures 48

of Lipase A gene with PKLAC2

plasmid using pKLAC2 forward

and lipase reverse as primers.

3.5.1 Lysate PCR analysis of E. coli 49

transformants (7-10) having the plasmids

with the inserts CALA using pKLAC2

forward and Lip A reverse as primers.

3.5.2 Analysis of plasmids extracted 50

by manual method from patches of

7, 8, 9, and 10 Top 10 E.coli

transformants

LIST OF TABLES

CHAPTER NO TITLE PAGE NO

1 1.1 Characeristics of CALA 14

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LIST OF SYMBOLS

CALA Candida antarctica Lipase A

⁰ C Degree celcius

DNA Deoxyribo Nucleic Acid

mg milligram

ml milliliter

rpm Rotation per minute

PCR Polymerase chain reaction

ul microlitre

LB Luria Bereni

kb kilobase pair

bp base pair

M molar

CaCl2 Calcium chloride solution

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CHAPTER 1

INTRODUCTION

1.1 LIPASES

Lipases (triacylglycerol hydrolases EC 3.1.1.3) belong to the family of

hydrolases which catalyze the hydrolysis of long chain triglycerides. These

enzymes are found in a vast species of plants, animals and microbes. Fungal lipase

is of much importance due to its significant application as biocatalyst. They are

used widely across food, flavor and biopharmaceuticals industry. Though

conventional techniques for commercial production of lipase incur higher costs,

this can be overcome by novel molecular biological techniques.

1.2 BIOCHEMISTRY OF LIPASES

Lipase family include structurally and functionally homologous group of

enzymes. The tertiary structure of lipases exhibits the hydrolase fold pattern

(Schrag and Cygler 1993). The lipase structure is composed of a core of upto eight

parallel beta strands, connected and surrounded by alpha helices. The active site is

formed by a catalytic triad consisting of a serine residue as the nucleophile,

histidine as base and aspartic (or glutamic) acid as the acidic residue. The active

site residues are placed inside a hydrophobic pocket termed as ‘nucleophilic

elbow’ and the pocket is covered by a lid like structure, composed of one or two

amphiphillic alpha helices. The activation of lipases requires the opening up of the

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pocket by the displacement of the lid and this process is known as interfacial

activation.

The catalytic mechanism of lipase hydrolysis consists of four subsequent

steps: i) the absorption and activation of the lipase at the interface between aqueous

and organic phase and then binding of the ester substrate within the hydrophobic

pocket; ii) in the second step, the nucleophilic oxygen of the serine side chain

attacks the carbonyl carbon atom of the ester bond leading to the formation of a

tetrahedral intermediate and this is stabilized by one or two hydrogen bonding with

amide nitrogen atom of the amino acid residues located in the region called as

‘oxyanion hole’. iii) The ester bond is then cleaved liberating an alcohol and leaves

behind the acyl-enzyme complex. In the last step, the acyl-enzyme is hydrolyzed,

when a water molecule (sometimes another alcohol) enters the active site, thereby

liberating the free fatty acid (or a new trans-ester with the alcohol) and the enzyme

is regenerated. Example of lipase catalysis mechanism is given in Figure 1.1.

Figure 1.1 Mechanism of lipase catalysis

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1.3 LIPASE ACTIVITY ASSAY

A wide range of assay protocols have been developed for the estimation of

lipase activity (Beisson et al. 2000). Based on the general triacylglycerol

hydrolysis reaction, the lipase activity is assayed by the release of either fatty acids

or glycerol from triacylglycerols or fatty acid ester. The various procedures used

for lipase analysis are summarized below.

i) PLATE ASSAY

Agar paltes supplemented with triacylglycerides such as tributyrin and

triolein are used in this assay. Lipolysis of triacylglycerides produces a clear

halo or colour change of Phenol Red / Victoria Blue / NileBlue Sulphate, or

fluorescence with Rhodamine B under UV light due to dye-Free fatty acid

complexation.

ii) TITRIMETRY

It involves potentiometric determination of free fatty acids liberated upon

hydrolysis by lipase. Simple titration by neutralization reaction of these free

fatty acids with NaOH to constant end-point pH can also be used to estimate

lipase.

iii) SPECTROPHOTOMETRY

Estimation of the lipase hydrolyzed yellow-colored p-nitro phenol at 420 nm

and 2,4-dinitrophenol at 360 nm gives the lipase activity .Also precipitation

of free fatty acids with calcium or copper and measurement at 500 nm gives

the lipase activity.

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iv) FLUORESCENCE

Lipolysis of fluorogenic substrates such as ω-linked pyrenic acyl-glycerol

derivatives gives lipase activity quantified in terms of increasing

fluorescence intensity with time.

1.4 REVIEW OF LITERATURE

1.4.1 Candida antarctica LIPASES

Among the widely used enzymes for biocatalytical purposes are the lipases

produced by different strains of genus Candida sp. In the late 1960’s the yeast

strain Candida antarctica was isolated from Lake Vanda in Antarctica and was

found to produce two different lipases (CALA and CALB) The two lipases of C.

antarctica were characterized and the amino acid and the DNA sequences

encoding these lipases were sequenced at Novo Nordisk A/S, by Patkar, Hoegh

and others. The lipase B was later crystallized and its structure was also

determined by Uppenberg et al. (1994).

C. antarctica lipase A and B were found to exhibit varying physio-chemical

characteristics. Lipase A is the most thermostable lipase known, being able to work

efficiently at >90°C. Such thermo stable enzymes are useful for industrial

applications such as pitch control in the paper industry, in the pulp and wax

industries and for asymmetric biocatalysis. The lipase B has become one of the

most prominent enzymes, in organic synthesis, especially for the kinetic resolution

of race mates. Currently, lipase B is the widely targeted enzyme for protein

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engineering so as to improve and optimize its substrate specificity and

enantioselectivity (Lutz 2004).

As both C. antarctica lipases have gained significant commercial

importance and as the expression levels in the native organism are too low,

recombinant over-expression is needed for the large-scale production of these

biocatalysts.

Table 1.1 Characeristics of CALA

Properties Value

Molecular Weight (kDa) 45

Isoelectric point (pI) 7.5

pH stability 6-9

Thermostability at 70° C 100[100]

1.4.2 INDUTRIAL APPLICATIONS OF LIPASES

The physical and chemical properties of Lipases that contribute to their

importance in industrial applications are i) stability in organic solvents ii) high

specificity iii) high enantio and regioselectivity and iv) no necessity for cofactors.

i) LOW CALORIE FAT PRODUCTION

Low calorie sunflower oil can be produced by inter-esterification with lipase

and behenic acid. Low calorie lipids are also obtained by interesterification

of tristearin with tricaprin or tricaprylin using an immobilized lipase.

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ii) MANUFACTURE OF COCOA BUTTER SUBSTITUTE

Cocoa butter which is used in confectionery industry has a high production

cost. Cocoa butter alternatives with similar flavor can be manufactured by

interesterification of less expensive fats like illipe fat, sal fat and shea butter.

iii) ACCELERATION OF CHEESE RIPENING

Lipases catalyze lipolysis in milk and therefore accelerating the cheese

ripening process.

iv) DETERGENT INDUSTRY

Lipolase, a lipase based proteolytic detergent has an optimal pH 10.5 – 11.0

and is active over a broad range of temperatures.

v) PHARMACEUTICAL INDUSTRY

Lipase is used in pharmaceutical industry as a biocatalyst as it avoids

isomerization, racemization, epimerization and rearrangement reactions in

chemical synthesis of drugs.

iv) RESOLUTION OF RACEMIC MIXTURES

The enantioselective nature of lipases is used in resolution of racemic

mixtures of molecules, thereby producing only one enantiomer in higher

concentration.

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1.5 E.coli MAINTANACE HOST

Genotype: TOP10F´: F'{lacIqTn10 (TetR)} mcrA Δ(mrr-hsdRMS-

mcrBC) Φ80lacZΔM15ΔlacX74 recA1 araD139 Δ(ara-leu)7697 galU galK

rpsL endA1 nupG

a) TOP10 F’ E.Coli strain is ideal for high-efficiency cloning and plasmid

propagation. They allow stable replication of high-copy number

plasmids. It has transformation efficiency greater than 1*109 /μg DNA.

b) hsdR for efficient transformation of unmethylated DNA from PCR

amplifications.

c) IrecA1 for reduced occurrence of non-specific recombination in cloned

DNA.

d) F´ episome carries the tetracycline resistance gene and allows isolation of

single-stranded DNA from vectors that have an f1 origin of replication.

1.6 K.Lactis EXPRESSION SYSTEM

Kluyveromyces lactis is a yeast commonly used in genetics research and

could potentially be used to produce pharmaceuticals or other compounds

Kluyveromyces lactis is a yeast species commonly used for genetic studies and

industrial applications.

Kluyveromyces lactis(formerly Saccharomyces lactis) is a yeast which has

the ability to assimilate lactose and convert it into lactic acid. Kluyveromyces lactis

and other organisms ie, Aspergillus niger var awamori and Escherichia coli K12

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are grown in fermenters to produce chymosin (rennet) on a commercial scale; this

rennet, which replaces the conventional form obtained from slaughtered animals, is

now widely used in cheese production.

ADVANTAGES:

a) High level and scaleable expression of recombinant proteins.

b) Rapid high cell density growth.

c) Simultaneous expression of multiple proteins possible.

d) No background gene expression during E. coli cloning steps.

e) Easy and fast cell transformation procedure.

f) No expensive antibiotics required.

g) Attractive commercial sublicensing.

REASONS FOR USING pKLAC2

• Variant of LAC4 promoter found in K. lactis

• No background transcription

• Even genes toxic to E. coli can be cloned

• Presence of multiple cloning sites

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Figure 1.2 pKLAC Vector Map

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CHAPTER 2

MATERIALS AND METHODS

2.1 HOST STRAINS

E. coli TOP10F’ – Maintenance Host

K. lactis – Expression Host

2.2 VECTORS

pKLAC2 vector system integrated in E. coli DH5α is used.

2.3 ANTIBIOTICS

Antibiotics are used for the selection of transformants. Here Ampicillin is

used. The working concentration of ampicillin is 100μg/ml which is used in the

selection process. Some bacteria exposed to penicillin survived because they

produced the enzyme β-lactamase that destroys penicillin’s structure. These

bacteria are known to contain antibiotic resistance gene.

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2.4 CULTURE MEDIUM

LB Medium (Luria Bertain Medium) .1000 mL of LB medium contains:

1000 mL deionized water

10 g Bactotryptone

5 g Bacto yeast

5 g NaCl several drops

5 M NaOH several drops

1 M HCl

2.5 GENE OF INTEREST

LIPA is the gene of interest that is isolated from Candida Antarctica.

2.6 POLYMERASE CHAIN REACTION

A Polymerase Chain Reaction (PCR) was done using gene specific

primers to amplify the gene of interest from the plasmid pPICZalpha B

REAGENTS REQUIRED

1. Master Mix (2X)

2. Forward primer

3. Reverse primer

4. Template DNA

5. Double distilled water

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REACTION CONTENTS

Template DNA (pPICZalpha B) 1.5 µl

lipA Xho1 forward primer 1 µl

lipA Xho1 reverse primer 1 µl

Double distilled water 6.5 µl

2X Master Mix 10 µl

REACTION VOLUME 20 µl

The reaction mixture for 20μl was prepared by taking water, DNA, master mix,

primer, & enzyme sequentially.

REACTION CONDITIONS

S.No Steps Temperature Duration

1 Initial denaturation 95˚C 5 min

2 Denaturation 95˚C 1 min

3 Annealing 60˚C 1 min

4 Extension 72˚C 2 min

5 Goto step 2 – 30cycles

6 Final extension 72˚C 10 min

7 Hold 4˚C

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2.7 AGAROSE GEL ELECTROPHORESIS

PRINCIPLE

Electrophoresis through agarose gel is the standard method used to separate,

identify and purify DNA fragments from 200 bp to 50 kb in length. When an

electric field is applied across the gel, the negatively charged DNA at neutral pH

migrates towards the anode. The rate of migration is determined by the number of

parameters such as molecular size of the DNA, agarose concentration,

conformation of the DNA (super helical circular, nicked circular and linear),

applied voltage direction of the electric field, composition of the electrophoresis

buffer.

REAGENTS REQUIRED

Running buffer (TEB 0.5X buffer) for 100 ml

Tris540mg,

EDTA46mg,

Boric acid275mg

pH 8.3

Gel loading dye

40% sucrose (w/v) in water, 0.25% orange G & 10% v/v (1X) TEB

Ethidium Bromide (EtBr)

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PROCEDURE

1. To the 40ml of 0.5 X TEB buffer, 320mg of agarose was added.

2. The solution was boiled for 50-60 seconds to dissolve the agarose.

3. When a bearable temperature was reached, 2.5μl of EtBr was added &

mixed

4. Properly.

5. The comb was placed on the gel tray in appropriate place.

6. The mixture was then poured into a gel tray containing comb.

7. It was allowed to solidify for 20 minutes.

8. The gel tray was kept into a gel tank containing 0.5 X TEB buffer. The

buffer

9. Level in a tank should be maintained above the gel tray.

10. The comb was then removed gently to avoid damage of the wells; the gel is

now ready for loading.

11. The DNA samples were loaded and the electrophoresis tank was connected

to the power pack & the voltage was set to 100V

12. The bands were observed under UV transilluminator.

2.8 RESTRICTION

The PCR product obtained is then restricted with Xho1 and

dephosphorylated for it to be used in ligation. Here the restriction is done with a

single enzyme Xho1.

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PRINCIPLE

Restriction endonucleases are enzymes that recognize & cut a specific

sequence of DNA. They are member of large group of enzymes called nucleases

which generally breaks the phosphodiester bond that link adjacent nucleotides in

DNA. Endonucleases cleave DNA at internal position. Restriction endonucleases

are categorized into three general groups (Types I, II and III) based on their

composition and enzyme cofactor requirements, the nature of their target sequence,

and the position of their DNA cleavage site relative to the target sequence. They

cut DNA at sites outside of their recognition sequence. Using ATP as energy

source, the enzyme move along the DNA molecule from recognition site to

cleavage site.

REAGENTS REQUIRED

1. Xho1 enzyme 1 µl

2. lip A gene 30 µl

3. Buffer 3 6 µl

4. 10X BSA 6 µl

5. Double distilled water 17 µl

REACTION VOLUME 60 µl

PROCEDURE

1. The reaction mixture for 40μl was prepared by taking water, DNA,

buffer, BSA solution and restriction enzymes sequentially.

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2. The mixture was incubated at 37˚C for 3hours for digestion to occur.

3. The digested sample was screened by 1 % Agarose gel electrophoresis

2.9 PURIFICATION

The restricted lip A gene PCR product was the purified before continuing on

to ligation.

REAGENTS REQUIRED

1. Sample ( gene)

2. Isopropyl alcohol (IPA)

3. QG buffer

4. Ethanol

5. PE buffer

6. MQ water

7. Columns

PROCEDURE

1. 80 µl of sample was added to 250ul of QG buffer and mixed well

2. 80 µl of IPA was then added to this mixture and mixed well

3. This is then poured onto a column and was centrifuged at 13000 rpm for 1

min.

4. The flow through from the column was discarded and then this step is

again repeated.

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5. 0.75ml of PE buffer and ethanol in the ratio of 1:3 was added to the

column.

6. Again centrifuge the column at 13000 rpm for 1 min.

7. Decant the flow through and dry spin the column.

8. The column is then placed in a new eppendorf tube and eluted it with 15 µl

MQ water. This is taken as elute1 (E1).

9. Again elute it with 15 µl MQ water and take it as elute2 (E2).

10. The E1 and E2 were run on 1% Agarose gel to confirm the purification of

gene.

2.10 STOCK PREPARATION: (30% GLYCEROL STOCK)

1. Add 300µl glycerol to empty eppendorf

2. Add 700 µl of sample (pKLAC2 DH5α) to the tube and mix thoroughly

3. Seal it with parrafin

2.11 INOCULATION

1. 3 µl of ampicillin was added to 3ml of LB medium in a test tube

2. 50 µl of pKLAC2 DH5α (glycerol stock) to the tube

3. Keep at 37˚C for overnight

2.12 PLASMID EXTRACTION

After restriction the restricted gene has to be ligated in the pKLAC2

DH5α maintenance host for the cloning process. For this the plasmid has to be first

extracted and then restricted so that the lipase A gene can be incorporated in it.

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REAGENTS REQUIRED

1. P1 buffer

2. P2 buffer

3. N3 buffer

4. QIA prep spin column

5. PB buffer

6. PE buffer

7. Ethanol

8. Double distilled water

PROCEDURE

1. Aliquot the sample (pKLAC2 DH5α) in test tube into 2 eppendorf tubes

2. Centrifuge at 4000rpm for 10min to pellet it

3. Add 250 µl of P1 buffer to the pellet and resuspend it

4. Vortex for 1min

5. Add 250 µl of P2 buffer and mix thoroughly by inverting the tubes 2 times

6. Add 300 µl N3 buffer and mix immediately and thoroughly by inverting the

tubes 4-6 times

7. Centrifuge for 10min at 13000 rpm

8. Apply the supernatant to the QIA prep spin column by decanting or pipetting

9. Centrifuge for 30-60sec and collect the flowthrough

10. Wash the QIA prep spin column by adding 0.5ml PB buffer and centrifuge

for 30-60sec. Discard the flowthrough

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11. Wash QIA prep spin column by adding 0.75ml (200 µl PE buffer + 800 µl

ethanol) of PE buffer and ethanol in ratio 1:4 and centrifuge for 30-60sec.

Discard the flowthrough

12. Dry spin the column

13. To elute the DNA , place the QIA prep column in a clean 1.5ml eppendorf

tube and add the following:

i. 70 µl water in column for 1 min and spin. Collect flowthrough

E1

ii. 50 µl water in column for 1 min and spin. Collect flowthrough

E2

iii. 30 µl water in column for 1 min and spin. Collect flowthrough

E3

The DNA is the confirmed by running the elutes (E1, E2 and E3) in a 1% agarose

gel.

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Figure 2.1 Plasmid Extraction

2.13 RESTRICTION

The plasmid obtained is then restricted with Xho1 and

dephosphorylated for it to be used in ligation. Here the restriction is done with a

single enzyme Xho1.

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Small scale restriction large scale restriction

Xho1 1 µl 1 µl

10xbuffer 2 µl 4 µl

10x BSA 2 µl 4 µl

pKLAC2 E1 8 µl 20 µl

Double distilled water 7 µl 11 µl

REACTION VOLUME 20 µl 40 µl

PROCEDURE

1. The reaction mixture for 40μl was prepared by taking water, DNA,

buffer, BSA solution and restriction enzymes sequentially.

2. The mixture was incubated at 37˚C for 2hours for digestion to occur.

3. The digested sample was screened by 1% Agarose gel electrophoresis.

2.14 DEPHOSPHORYLATION

Antarctic Phosphatase catalyzes the removal of 5´ phosphate groups from

DNA and RNA. Since phosphatase-treated fragments lack the 5´ phosphoryl

termini required by ligases, they cannot self-ligate. This property can be used to

decrease the vector background in cloning strategies.

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REACTION MIXTURE

Antarctic Phosphatase enzyme buffer 1.5 µl

Antarctic Phosphatase enzyme 1.0 µl

pKLAC2 elute 1 15 µl

Incubate for 15 min at 37°C

Inactivate immediately at 65°C for 5 min.

The Antarctic Phosphatase has 90% efficiency that is out of 10 DNA molecules

only 9 are dephosphorylated. Antarctic Phosphatase buffer is added, so that overall

75% of plasmid DNA molecules are dephosphorylated.

Applications of antarctic phosphatase

Removing 5´ phosphoryl groups from nucleic acids

Preparing templates for 5´end labeling

Preventing fragments from self-ligating

Removal of dNTPs and pyrophosphate from PCR reactions

2.15 PURIFICATION

The restricted plasmid was the purified before continuing on to ligation.

REAGENTS REQUIRED

Sample (plasmid)

Isopropyl alcohol (IPA)

QG buffer

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Ethanol

PE buffer

MQ water

Columns

PROCEDURE

1. 80 µl of sample was added to 250 µl of QG buffer and mixed well

2. 80 µl of IPA was then added to this mixture and mixed well

3. This is then poured onto a column and was centrifuged at 13000 rpm for 1

min.

4. The flow through from the column was discarded and then this step is again

repeated.

5. 0.75ml of PE buffer and ethanol in the ratio of 1:3 was added to the column.

6. Again centrifuge the column at 13000 rpm for 1 min.

7. Decant the flow through and dry spin the column.

8. The column is then placed in a new eppendorf tube and it is eluted with 15

µl MQ water. This is taken as elute1 (E1).

9. Again elute it with 15 µl MQ water and take it as elute2 (E2).

10. The E1 and E2 were run on 1.5% Agarose gel to confirm the purification of

plasmid.

2.16 LIGATION

PRINCIPLE

Ligation process involves the formation of four phosphodiester bonds i.e

two at each end of the molecule such bonds can be formed. The generation of

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phosphodiester bond between neighbouring 3’O ُ H end and 5’P ُ ends of double

stranded DNA chain is catalyzed by DNA ligase and they require coenzymes like

NAD+ & ATP.

Figure 2.2 A pictorial example of how a ligase works (with sticky ends)

34

Figure 2.3 A simple mechanism representing the role of DNA Ligase

35

REAGENTS REQUIRED

Reaction Volume: 20µl

pKLAC2 2 µl

Lip A gene 15 µl

10X buffer 2 µl

DNA ligase 1 µl

Doubled distilled water 0 µl

Vector control

pKLAC2 2 µl

Lip A gene insert 0 µl

10X buffer 2 µl

DNA ligase 1 µl

Doubled distilled water 15 µl

PROCEDURE

1. The ligation mixture for 20μl was prepared by taking water, insert (Lipase A

gene), and vector digest & ligase enzyme sequentially.

2. The mixture was incubated at 16˚C for overnight

3. The ligated sample was screened by agarose gel electrophoresis.

The ligated cells were then transformed in E. coli and then cloned.

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2.17 COMPETENT CELLS PREPARATION AND TRANSFORMATION

PRINCIPLE

E.coli cells take up only limited amount of DNA under normal

circumstances E.coli cells when soaked in an ice cold salt solution were more

efficient in DNA uptake than by unsoaked cells. CaCl2 causes the DNA to

precipitate onto the outside of the cells or perhaps the salt is responsible for some

kind of change in cell wall that improves DNA binding. E.coli cells and plasmid

interact productively in an environment of Ca ions & low temperature (0-5˚C). The

calcium ions destabilize the cell membrane and a calcium phosphate DNA

complex is formed which adheres to the cell surface and is resistant to DNases.

The DNA is taken up during a heat shock step where the cells are exposed briefly

to a temperature at 42˚C.

REAGENTS REQUIRED

1. Overnight bacterial culture

2. 0.1M ice cold CaCl2 solution

3. Plasmid suspension

4. LB medium

5. LB/amp+ agar plates

37

PROCEDURE

1. From overnight inoculated culture of DH5α host 1ml of culture was

inoculated in 50ml LB medium

2. After 1 hour 45 min the flask was taken and kept at 4˚C

3. Transfer the culture in 2 centrifuge tubes equally and keep it in ice

4. Then centrifuge at 4000 rpm for 10 min

5. Discard the supernatant. Add small amount of CaCl2 solution and

resuspend the pellet by tapping. Then add CaCl2 solution to 3/4th of the

tube

6. Incubate for 30 min at 4˚C

7. Centrifuge at 4000rpm for 10 min

8. Discard supernatant. Add small amount of 0.1M CaCl2 solution and

resuspend the pellet by tapping. Then add CaCl2 solution to 1/4th of the

tube

9. Incubate for 20 min at 4˚C

10. Centrifuge at 4000 rpm for 10 min

11. Discard the supernatant. Add 400 µl of CaCl2 solution to one tube and

resupend it. Pool it to the other tube and add a little more according to

thickness of the pellet

12. Incubate for 25-30 min at 4˚C

38

2.18 TRANSFORMATION

PRINCIPLE

In molecular biology, transformation is the genetic alteration of a cell

resulting from the uptake, genomic incorporation, and expression of environmental

genetic material (DNA). Transformation occurs most commonly in bacteria, both

naturally and artificially, and refers to DNA taken up from the environment

through their cell wall. Bacteria that are capable of being transformed are called

competent. New genetic material can also be transferred to cells through

conjugation or transduction. Conjugation involves cell-to-cell contact between two

different bacterial cells, with the DNA being transferred from one bacterial cell to

the other. In transduction, viruses called bacteriophages inject the foreign DNA

into their host. Introduction of foreign DNA into eukaryotic cells is usually called

"transfection". Transformation is also used to describe the insertion of new genetic

material into nonbacterial cells including animal and plant cells.

ARTIFICIAL COMPETENCE

Artificial competence is not encoded in the cell's genes. Instead it is induced

by laboratory procedures in which cells are passively made permeable to DNA,

using conditions that do not normally occur in nature. Calcium chloride

transformation is a method of promoting competence. Chilling cells in the presence

of divalent cations such as Ca2+ (in CaCl2) prepares the cell membrane to become

permeable to plasmid DNA. Cells are incubated on ice with the DNA and then

briefly heat shocked (e.g. 42 °C for 30–120 seconds), which causes the DNA to

enter the cell. This method works very well for circular plasmid DNAs. An

39

excellent preparation of competent cells will give ~108 colonies per microgram of

plasmid. A poor preparation will be about 104/μg or less. Good non-commercial

preps should give 105 to 106 transformants per microgram of plasmid.

Figure 2.4 Artificial Transformation

PROCEDURE

1. 100 µl DH5α competent cells + 7 µl pKLAC2 lip A

100 µl DH5α competent cells + 7 µl vector (pKLAC2)

100 µl DH5α competent cells

2. Keep it in ice for 30 min

3. Then give heat shock at 42˚C for 90 sec

4. Then again place it in ice for 10-15 min

40

After the above process the cells are to be grown in LB/amp+ (for negative

selection) agar plates in 37˚C for 16 hrs and then the plates are checked for growth

of transformed colonies.

2.19 PATCHING

After the plates are checked for the presence of colonies they are to be

patched in new LB/amp+ agar plates. This is done in the following way.

1. Check the plate for presence of colonies

2. Take a loop and with it take a colony form the transformed plates and

streak on a new LB/amp+ agar plates.

3. Mark the streaked places and allow it grow overnight

4. After 12 hours check for the growth of colonies.

2.20 LYSATE PCR

Lysate PCR is necessary for conformation of the presence of plasmid

in the transformed colonies.

REAGENTS REQUIRED

2X Master Mix 55 µl

pKLAC2 promoter forward primer 5.5 µl

pKLAC2 TT reverse primer 5.5 µl

10 colonies + 1 vector control 11 µl

Double distilled water 33 µl

REACTION VOLUME 110ul

41

PROCEDURE

1. Take the 10 colonies from the patched and suspend it in 50 µl distilled

water

2. Heat at 100˚C for 10 min

3. Immediately place it in ice for 5 min

4. Centrifuge at 10000 rpm for 2 min and take only the supernatant for the

PCR

5. The PCR reaction was done ad explained before

2.21 CONFIRMATION PCR:

A confirmation PCR is done to check whether the required gene of interest is

inserted into the plasmid. This PCR is done to all the positively screened patches

(7, 8, 9 and10).

Negative control: pKLAC 2

Test : patches 7-10

2x MM : 25 µl

LipA forward : 2 .5 µl

KLAC TT reverse : 2 .5 µl

Template : 5 µl

Double distilled water : 15 µl

REACTION VOLUME : 50 µl

Here the template includes patch 7, 8,9,10 and a negative control.

42

Positive Control : pPICZ alpha B lipA

Negative control : pKLAC 2

Test : patches 7-10

2x MM : 30 µl

LipA forward : 3 µl

Lip A reverse : 3 µl

Template : 6 µl

Double distilled water : 18 µl

REACTION VOLUME : 60 µl

Here the template includes patch 7, 8,9,10, a negative control and a positive

control.

43

CHAPTER 3

RESULTS AND DISCUSSION

3.1. PCR AMPLIFICATION OF C.antartica LIPASE A ENCODING GENE

The PCR amplification of Lipase A was carried out using forward primer

with the Xho1 restriction enzyme site and reverse primer with Xho1 restriction

enzyme site. The cloning of lipases in the vector pKLAC2 was designed by

utilizing the XhoI site upstream of Kex2 cleavage site in vector so as to facilitate

the native expression of the recombinant protein. The forward primers also had the

sequences of the Kex2 cleavage sites downstream of the Xho I restriction site and

following this were the first 18 bases of the mature lipase A coding sequences. The

utilization of Xho I restriction site and Kex2 cleavage site enabled the expression

of recombinant lipase with their native N-terminus, after the cleavage of the signal

sequence from the expressed proteins by Kex2 proteinase. The reverse primers

utilized the ending 20 bases which code for the C-terminal regions of the lipases A

along with its stop codon.

CALAf - 5’-CCGCTCGAGAAAAGAGCGGCGCTGCCCAACCCC-3’

CALAr - 5’-CTAGCTCGAGCTAAGGTGGTGTGATGGGGC-3’

XHO1 RESTRICTION SITE

XHO1 RESTRICTION SITE

KEX2 CLEAVAGE SITE

44

Agarose gel electrophoresis of PCR amplified lipase A gene is shown in the figure

3.1.1

Figure 3.1.1 Analysis of PCR amplified lipase A gene

4 – 1 kb marker

2 – PCR amplified Lipase A gene from CAL A (1µl)

3.2 RESTRICTION AND PURIFICATION OF LIPASE A GENE

The PCR amplified Lipase A gene was restricted by Xho1 restriction

enzyme after three hours incubation. The restricted sample of Lipase A gene was

purified to remove excess salts and to increase the lipase A gene concentration.

The Agarose gel electrophoresis of purified samples – elute 1 and elute 2 of Lipase

A gene is shown in the figure 3.2.1.

1.4 kb

1 2 3 4 5

45

Figure 3.2.1 Analysis of purified samples of Lipase A gene

1 – 1 kb marker

2 – First elution of Purified sample of lipase A gene (0.5 µl)

4 – Second elution Purified sample of Lipase A gene (0.5 µl)

3.3 pKLAC2 – EXTRACTION AND RESTRICTION

Agarose gel electrophoresis of the pKLAC2 plasmid extracted is shown in

the figure 3.3.1. The plasmid extraction was carried out by standard kit method.

1 2 3 4

1.4 kb

46

Figure 3.3.1 Analysis of pKLAC2 plasmid extracted by standard Kit method

1 – 1 kb marker

4 – First elution sample of pKLAC2 Plasmid

5 – Second elution sample of pKLAC2 Plasmid

Plasmid extracted was restricted with Xho1 restriction enzyme. pKLAC2

plasmid was completely restricted after two hours incubation. Agarose gel

electrophroesis of restricted plasmid is shown in the figure 3.3.2. The pKLAC2

plasmid which was restricted with single enzyme xho1 was dephosphorylated to

prevent self-ligation of sticky ends. Antarctic Phosphatase was used to

dephophorylate pKLAC2 plasmid. Antarctic Phosphatase catalyzes the removal of

5´ phosphate groups from DNA and RNA. Since phosphatase-treated fragments

lack the 5´ phosphoryl termini required by ligases, they cannot self-ligate. This

property can be used to decrease the vector background in cloning strategies.

1 2 3 4 5 6 7

47

Figure 3.3.2 Analysis of restricted pKLAC2 plasmid

1 - Uncut pKLAC2 plasmid

2 - Restricted pKLAC2 plasmid

3.4 CLONING

The restricted lipase A gene was ligated with T4 ligase enzyme

Two Ligation mixtures containing lipase A gene and PKLAC2 vector in the

molar ratios of 3:1 and 5:1 were prepared and incubated at 16°C for 16 hours.

Agarose gel electrophoresis of two the samples is shown in the figure 3.4.1

Both the plasmid and lipase A were restricted using single restriction

enzyme Xho1. This kind of cloning is categorized as unidirectional cloning.

1 2

48

Because of this, the ligation mixtures were confirmed for proper orientation

through PCR using pKLAC2 forward and lipase A reverse as primers.

Figure 3.4.1 Analysis of ligation mixtures of Lipase A gene with PKLAC2

plasmid using pKLAC2 forward and lipase reverse as primers.

1 – 1 kb marker

2 – First ligation sample (1 µl)

3 – Second ligation sample (1 µl)

3.5 TRANSFORMATION

pKLAC2 vector with lipase A gene insert was transformed into E. coli TOP

10 F’ strain by calcium chloride method and selected on LB medium with

amphicillin.. Among the transformants ten colonies for lipase A gene were

analyzed by patching the colonies on fresh LB agar plates with amphicillin. And

lysate PCR analyses of all these transformants were done. The plasmids of the

positive transformants were confirmed for correct orientation of inserts in the

vector and that screening of transformants were carried out using the vector

1 2 3 4

1.8kb

49

forward(pKLAC2 forward) and the insert reverse(lipase A gene reverse) primers.

The presence of the lipase A gene inserts in the Patch 7, 8, 9, and 10 were

confirmed. A total of 4 positive transformants, having the plasmid with CALA

inserts were obtained.

The positive E. coli TOP 10 F’ strain transformants 7, 8, 9 and 10 were

cultured overnight in LB medium with amphicillin and the plasmid DNA was

extracted using manual methods. The plasmids were confirmed for the correct

orientation of the inserts in the vector by PCR using the vector forward (pKLAC2

forward) and the insert reverse (lipase A reverse) primers. Agarose gel

electrophoresis of these analyses is shown in figures 3.5.1. Here the genomic DNA

was used as positive control and the vector without insert was used as the negative

control.

Figure 3.5.1 Lysate PCR analysis of E. coli transformants (7-10) having the

plasmids with the inserts lipase A gene using pKLAC2 forward and Lip A

reverse as primers.

1 – Mol wt marker (1 kb)

2, 3, 4 & 5 – positive E. coli transformants having lipase A gene insert (1µl)

6 – Negative control (1µl)

1 2 3 4 5 6 7

1.8 kb

50

The plasmid extracted from the positive transformants 7, 8, 9 and 10 by

manual method are shown in figure 3.5.2. Due to manual method of extraction,

contamination was there as shown in the way in the figure.

Figure 3.5.2 Analysis of plasmids extracted by manual method from patches

of 7, 8, 9, and 10 Top 10 E.coli Positive transformants

2, 3, 4 and 5 – plasmids extracted from Patches of 7, 8, 9 and 10 positive E.coli

positive transformants(1.5µl).

1 2 3 4 5 6

51

CHAPTER 4

CONCLUSION

The CALA gene from Candida antarctica was successfully amplified by

PCR using forward and reverse primers with Xho1 restriction site. The lipase A

gene was purified and ligated with pKLAC2 plasmid restricted with Xho1

restriction enzyme. The recombinant plasmid pKLAC2/CALA was constructed

successfully. The recombinant plasmid was successfully transformed into E.coli

TOP 10F’ strain by chemical transformation. The transformant E.coli cells were

screened to verify successful transformation of recombinant plamid through Lysate

PCR.

The recombinant plasmid pKLAC2/CALA from the positive transformant

E.coli TOP10F’ cells can be linearised and transformed into the expression host

K.lactis for further expression studies. K.lactis is an ideal host for expressing of

heterologous proteins.

52

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