CLONING AND EXPRESSION OF TAQ POLYMERASE IN TOP10 E.Coli COMPETENT CELLS

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Cloning and expression of Taq polymerase in

top10 E. coli competent cells

Sanghamitra Kumar

Central University of Tamil Nadu, Tamil Nadu

Guided by

Dr. D. Karunagaran

Department of Biotechnology, Indian Institute of Technology Madras, Chennai, Tamil Nadu

600036

1. Introduction

Thermostable DNA polymerase is a very important enzyme for molecular biological studies such

as DNA amplification and DNA sequencing by the polymerase chain reaction (PCR). Taq DNA

polymerase is an enzyme obtained from a heat stable bacterium called T. aquaticus having a

molecular weight of about 6.6×10

–9.4×10

Daltons. T. aquaticus is a bacterium that lives in hot

springs and hydrothermal vents.

Taq polymerase was identified as an enzyme able to withstand the protein-denaturing conditions

(high temperature) required during PCR. Taq's temperature optimum for activity is 75–80°C, with

a half-life of 9 min at 97.5°C, and can replicate a 10 base pair strand of DNA in less than 10 s at

72°C.

Taq DNA polymerase catalyzes the incorporation of dNTPs into DNA. It requires a DNA template,

a primer terminus and the divalent cation Mg++. Taq polymerase contains a polymerization

dependent 5'-3' exonuclease activity. It does not have a 3'-5' exonuclease and thus no proof reading

mechanism. The thermostable properties of Taq DNA polymerase from Thermus aquaticus have

contributed greatly to the yield, specificity, automation, and utility of the polymerase chain

reaction method for amplifying DNA. Taq polymerase is widely used enzyme for DNA

amplification in PCR techniques and highly applicable in molecular biology and biotechnology.

Taq gene was amplified from the genomic DNA of the organism Thermus aquaticus and

cloned into pTaq plasmid. The Top 10 Escherichia coli were transformed using the recombinant

containing the gene of interest and the clones of right orientation were selected and followed

by protein induction.

Unlike regular techniques of visualising gel like Coomassie Brilliant Blue dye and Silver Staining,

I have improvised it by using chloroform which I have discussed later in the protocol below. I

came across this easy and rapid technique of using Chloroform or any trihalogen compound like

TCE in a paper by C. Ladner cited below.

2. Materials and methods

2.1. Amplifying Taq polymerase gene and cloning of gene in plasmid

A 2.6 Kb fragment containing the whole T. aquaticus DNA polymerase gene was prepared by PCR

amplification with the T. aquaticus genomic DNA using primers forward-primer and the reverse-

primer creating unique restriction sites. The fragment was ligated in a vector. This vector is

designated as pTaq plasmid and transformed into Top 10 E. coli cells.

We take a plasmid and clone it with our gene of interest. Next we transform into bacterial cells

with specific antibiotic resistance and grow them in the same antibiotic containing medium. Cells

that grow in the ampicillin media are cultured and grown.

Now amongst them, some contain our cloned gene of interest, while others do not. So we make

each plasmid undergo restriction digestion and check for our cloned gene. Bacteria which contain

the same are being separated and cultured.

From these bacteria again plasmid is being isolated using Midiprep plasmid isolation by alkaline

lysis method.

Plasmid isolation (by alkaline lysis method): Pelleted bacterial cells are resuspended and

subjected to SDS/alkaline lysis to liberate plasmid DNA. The resulting lysate is neutralized

allowing denatured plasmid DNA to reanneal while cell debris such as proteins, chromosomal

DNA and SDS precipitate out of solution. The resulting precipitate is pelleted by centrifugation

and the supernatant containing plasmid DNA is loaded onto the purification column. The high salt

concentration of the lysate creates the appropriate conditions for plasmid DNA binding to the silica

membrane in the spin column. The adsorbed DNA is washed to remove contaminants and eluted

with the elution buffer.

• Inoculate 100ml culture with a glycerol stock with appropriate antibiotic like ampicillin

and allowed to grow overnight in a shaking incubator (180 rpm) at 37°C.

• Spin down the culture in autoclaved tubes at 6000 rpm for 6 min.

• The supernatant is discarded and pellet is resuspended in 3 ml of Solution 1 followed by 6

ml of Solution 2. Keep on ice for 5 min and then dissolve in 4.5 ml of Solution 3. Keep in

ice for 10 min after that.

Solution 1:

500 Mm glucose –10ml

500 Mm EDTA (pH-8) –2ml

1M Tris (pH-8) –2.5ml

Water –85.5ml

Solution 2:

0.2NaOH

1% SDS

Solution 3:

3M KOAc

60 ml 5M potassium acetate (49.07 g in 100 ml water)

11.5 ml glacial acetate

28.5 ml water

• Centrifuge at 12000 rpm for 15 min.

• Take supernatant and add equal amount of chilled isopropanol.

• Centrifuge at 12000 rpm for 15 min.

• Discard supernatant and to pellet, add 70%chilled ethanol to wash it and air dry it on tissue

paper. But it shouldn’t be over dried.

• The pellet is then dissolved in 1.5 ml water.

• Transfer it into two tubes 750 µl each.

• Add equal amount of 5M LiCl2 (750 µl) which precipitates the RNA.

• Incubate on ice for 30 min.

• Centrifuge for 12000 rpm for 10 min.

• Now discard the pellet and to supernatant add equal volume of isopropyl alcohol

• Centrifuge again for 12000 rpm for 10 min.

• Discard the supernatant. Wash pellet with 70% ethanol and dry it.

• Add 1 ml of RNAse buffer in each and rinse with pipette.

• Incubate it in hot oven for 1–1.5 h.

• Next add equal amount of isoamyl-chloroform-phenol. This step extracts proteins from

nucleic acid and denatures proteins.

• Centrifuge at 14000 rpm for 15 min.

• To aqueous phase, add double amount of alcohol and one-tenth of sodium acetate

• Centrifuge at 12000 rpm for 15 min.

• Discard the supernatant. Wash pellet with 70% ethanol and dry it.

• Resuspend the pellet in sterile double distilled water or TE buffer and store at –20°C.

• This pellet contains the purified plasmid.

2.2. Competent cells

E. coli top10 competent cells: We used TOP10 E. coli cells which are ultra competent. They are

provided at a transformation efficiency of 1 × 10 9 cfu/μg supercoiled DNA and are ideal for high-

efficiency cloning and plasmid propagation. They allow stable replication of high-copy number

plasmids. The genotype of TOP10 E. coli Cells is similar to the DH10B TM strain genotype.

2.3. Transformation

Transformation is the process in which these competent cells take up the foreign pTaq plasmid.

Transformation of bacteria with plasmids is important not only for studies in bacteria but also

because bacteria are used as a mean of both storing and replicating plasmids. Because of this reason

most of plasmids contain a bacterial origin and an antibiotic resistance gene marker.

• The pTaq plasmid (0.3 μl) was added to ultracompetent cells which were stored in –80°C

and were kept on ice for 2 min.

• Next heat shock was given by keeping the tubes in a hot water bath (42°C) for exact 90 s.

Heat shock is a crucial step

• Rapidly tubes were transferred to an ice bath and let it cool for 2–3 min.

• LB broth media (900 μl) was added to each tube and cultures were warmed to 37°C in a

water bath and later transferred to a shaking incubator set at 37°C (180 rpm).

• Plate 100 μl of this media on LB plates prepared before containing antibiotic, ampicillin.

These LB plates are prepared beforehand.

2.3.1. Primary culture

• 5 μl of ampicillin is added to 5 μl of LB broth in a tube.

• The LB plates are scrutinised and a single colony is identified.

• We use a teaser to add the bacteria from the plate to the tube containing ampicillin.

• In this step we make use of a control by taking another similar broth tube with ampicillin

but no bacteria.

• These two tubes are incubated overnight.

• Next morning it is observed that one of the tubes turn turbid, i.e., the one containing

bacteria, while the control does not.

• Again 100 μg/ml ampicillin is added to 100 ml broth without touching the media.

• This culture is called the primary culture.

2.3.2. Secondary culture

• 1% of primary culture bactria is aliquoted. This is the secondary culture.

• 1% of 100 ml is 1ml and hence 1 ml is aliquoted.

3. Induction of protein synthesis, gene expression in bacteria with IPTG

Control of the pTaq expression system is accomplished through the lac promoter and operator.

Before target gene can be transcribed, T7 polymerase must be present. The gene on the host cell

chromosome usually has an inducible promoter which is activated by addition of Isopropyl-beta-

thio galactopyranoside (IPTG). This molecule, IPTG, displaces the repressor from the lac operator.

Since there are lac operators on both the gene encoding T7 polymerase and target gene, IPTG

activates both genes. Therefore, when IPTG is added to the cell, the T7 polymerase is expressed,

and quickly begins to transcribe target gene which is then translated as desired protein. IPTG works

to displace a lac repressor since IPTG is an analogue of lactose. The lac genes express enzymes

which are involved in the breaking down of lactose, and therefore, the presence of lactose [or its

analogue] would trigger the initiation of transcription of lac genes.

• The secondary culture was placed on a shaking incubator at 37°C for 3 h.

• The optical density of the culture was measured time to time.

• When the O.D. reached 0.6, expression of recombinant protein was induced by 0.52 mM

IPTG to the growing culture for 3 h.

• The culture was kept overnight. No addition of inducer was used as negative control

experiment.

4. Protein extraction and purification

• The culture is centrifuged at 6000 rpm for 15 min.

• The supernatant is discarded .

• To the pellet, 5 ml of sonication buffer is added. This is mixed well and kept for storage

overnightat –80°C.

Sonication buffer:

50 mM trisHCl(pH-8.0) – 2.5 ml

300 mM NaCl –3.75 ml

1 mM EDTA –0.1 ml

1 mM PMSF –0.5 ml

• Next day, we thawed it at room temperature and went for sonication for 2 min. In this

stage the cells undergo subjection to periodic ultrasound and hence get lysed.

[ Amplitude: 36% Pulse on/off : 3 s]

• The sonicated liquid is aliquoted into 2 ml tubes and heated at 90°C for 30 min.

• After that, tubes were taken out and kept on ice for 10 min.

• The tubes are centrifuged at 12000 rpm for 10 min.

• The pellet is discarded and supernatant collected.

• This supernatant contains the Taq polymerase enzyme protein.

• This protein is further purified and concentrated by the process of dialysis.

5. SDS-PAGE

SDS PAGE is the type of electrophoresis in which protein molecules designated negative charge

by the detergent sodium dodecyl sulphate separate according to their molecular weight under

elcetric field.

The Laemmli SDS-PAGE system can be considered a 3-component system. The stacking and

running (resolving) gels have different pore sizes, ionic strengths and pHs. The third component

is the electrophoresis buffer (25 mM Tris, 192 mM glycine,, 0.1% SDS, pH ~8.3), which contains

large amounts of glycine. The ionization state of the glycine is critical to the separation. At neutral

pH, glycine is a zwitterion, with a negatively charged carboxyl group and apositively charged

amino group. The pKa of the amino group is 9.6, considerably higher than the pH of the chamber

buffer.Consequently, very little glycine has a negative charge in thechamber buffer or stacking gel,

and significant ionization doesnot occur until the glycine enters the more alkaline pH 8.8

environment of the running gel.

• The resolving gel is prepared and cast into the Bio-Rad electrophoresis setup.

• The gel is allowed to polymerise.

• Stacking gel.

• The comb is placed and gel is allowed to solidify.

• The comb is taken out and running buffer is added to the wells.

• Next the buffer is poured out and to each well the extracted and purified Taq polymerase

protein is loaded in concentrations of 10–90 μl. The first well is added with the protein

ladder.

• Add loading dye to each well.

• Run the gel for 1–2 h at 120 V.

Running buffer:

• Glycine

• Tris base

• SDS

• double distilled water

Resolving gel

Stacking gel

Water

4 ml

3.4 ml

Solvent

30% polyacrylamide

3.3 ml

0.83 ml

Helps polymerisation

1.5M Tris HCl

2.9 ml

0.63 ml

Buffer

10% SDS

0.1 ml

0.05 ml

Allocates negative charge

10% APS

0.1 ml

0.05 ml

Initiates polymerisation

10% TEMED

0.004 ml

0.005 ml

Catalyses polymerisation