Tabashir Chowdhury ID: 211219136

BIOL3140
Advanced Biochemistry and Molecular Genetics Laboratory

Laboratory Report 2
Cloning the Alpha amylase gene from Bacillus licheniformis by PCR

TA: Mez

Date of Submission: 11/11/11

Tabashir Chowdhury ID: 211219136

Abstract
The goal of this experiment was to clone the amyE gene from B.licheniformis into E.coli. Polmerase Chain Reaction (PCR) was used to amplify a specific region of B.licheniformis chromosomal DNA that contains the alpha amylase gene. A plasmid pET-15b is used as a vector to clone this region into E. coli. Both the vector and the PCR amplified DNA are cleaved at specific using the restriction enzymes Nde1 and BamH1 and then ligated using T4 DNA ligase. The recombinant plasmid DNA thus formed is introduced into competent E.coli bacterial cells by Transformation using heat shock treatment. The cloning vector pET-15b contained an ampicillin resistance gene Ap (46435500) that was used to select for transformed colonies, by growing the culture in an ampicillin medium. The PCR cloning of the amyE gene was verified via colony PCR followed by gel electrophoresis, which showed a 1.6 Kb DNA fragment corresponding to the alpha amylase gene. An amylase positive plasmid pAMY8 was used as a positive control, which produced an identical band at 1.6 Kb thus confirming a successful cloning of the amyE gene.

Tabashir Chowdhury ID: 211219136

Introduction
The purpose of this experiment is to clone the alpha amylase gene from Bacillus licheniformis using polymerase chain reaction, and inserting the gene into E. coli by transformation using a vector plasmid pET-15b. This allows the expression of alpha amylase protein by the E. coli. -Amylase is an enzyme that hydrolyses alpha-bonds of large alpha-linked polysaccharides such as starch and glycogen, yielding glucose and maltose (1). It is the major form of amylase found in humans and other mammals as well as in many strains of Bacillus. The alpha amylase enzyme is of particular interest in industry because of its remarkable thermostability (2) and thus plays a major role in the starch processing industry. The alpha-amylase expressed by the B. licheniformis amyE gene has a lower optimum temperature at 430C (3), which allows for a lower energy input and is therefore much more economically feasible and desirable for industrial use. In this experiment the alpha amylase gene from B. licheniformis was amplified using PCR cloning techniques after high molecular weight DNA was extracted from B. licheniformis cells using a phenol: chloroform: isoamyl alcohol (25:24:1) mixture (4). The amyE gene was then inserted into competent E. coli cells by DNA transformation via an expression vector pET-15b. Successfully transformed colonies were selected by their resistance to ampicillin acquired from the plasmid vector pET-15b. A colony PCR performed on the transformed E.coli cells followed by agarose gel electrophoresis confirmed the presence of the amyE gene in the E.coli DNA.

Tabashir Chowdhury ID: 211219136

Materials and Methods

All the materials used and the procedures followed were done according to the protocols listed in the BIOL 3140 Laboratory manual, Biotechnology: DNA to protein- A project in Molecular Biology by Theresa Thiel, et al. 2002. However, several changes were made during the course of the experiment. The changes to the protocol are listed in the BIOL 3140 Lab 3 supplementary sheet provided during the experiment. The PCR cleanup reactions were carried out according to the Qiagen PCR reaction cleanup kit manual provided during the Lab.

Tabashir Chowdhury ID: 211219136

Results
Chromosomal DNA from B. licheniformis was isolated. An agarose gel electrophoresis was then carried out using the extracted DNA sample to determine purity to and ensure a high molecular weight DNA. Figure 1 illustrates the results of the gel electrophoresis. Lane 1 contains the 1Kb DNA ladder, and lane 6 contains our sample DNA. The presence of a single distinct band indicates that the DNA extracted is very pure. Using the DNA ladder we can determine that the molecular weight of the sample is approximately 8Kb. The amount of DNA in the sample was estimated by taking absorbance readings at 260 nm and 280 nm. DNA absorbs maximally at 260 nm while proteins absorb maximally at 280 nm (4). A reading of 1 at 260 nm corresponds to a concentration of about 0.5 g/l. A hundred fold dilution of our DNA sample produced an absorbance reading of 0.256 at 260 nm and 0.127 at 280 nm. The concentration of DNA in the original sample was calculated to be 12.8 g/l. The ratio between the absorbance readings at 260 nm and 280 nm provides an estimate of the purity of the sample. Pure preparations of DNA samples have a A260/A280 ratio of 1.8 to 2.0 (4). Our sample had a ratio of 2.02, which confirms the purity of the sample. The concentration of a 20 fold diluted sample was 0.6g/l. A PCR reaction was performed using the B. licheniformis chromosomal DNA as a template. The PCR product and the vector pET15b were digested with Nde1 and BamH1 restriction enzymes. An agarose gel electrophoresis was performed with the double digested pET15b vector and the uncut pET15b vector. Figure 2 illustrates this gel image. Lane 1 contains the 1 Kb DNA ladder, lane 2 contains the double digested

Tabashir Chowdhury ID: 211219136 pET15b vector, with of 1.6 kb and lane 4 contains the uncut circular plasmid (pET15b) with a molecular weight of 5Kb

Figure 1 The agarose gel containing the B. licheniformis cDNA isolations. Lane one contains 1 KB ladder. lanes 2-6 contains the chromosomal DNA from the different groups 1 through 5. Lane 6 contains our DNA sample. The single band is observed approximately along the same line as the 8 KB band in the 1 kB DNA ladder.

Tabashir Chowdhury ID: 211219136

Figure 2 The agarose gel shows the vector pET-15b digested with Nde1 and BamH1, and the uncut circular plasmid. Lane 1 contains the 1Kb DNA ladder, lane 2 contains the double digested vector pET-15b, and lane 4 contains the undigested vector pET-15b

Tabashir Chowdhury ID: 211219136

Figure 3The standard curve for figure 2 restriction digest of the plasmid pET-15b constructed using the log of molecular weight in KB of the 1 Kb DNA ladder against the absolute distance migrated by DNA in the gel.

Table 1 The Date from the gel containing the digested pET-15b plasmid vector. The table contains the log of molecular weight in Kb of each band in the 1 Kb ladder in lane 1 of the gel, and the absolute distances travelled by the respective bands in the gel.

Log of Molecular Weight Kb 4 3.903 3.7782 3.698 3.602 3.477 3.301 3.1761 3 2.699

Absolute distance (mm) 11 12 13 14 15.5 16 17.5 20 23 27

Tabashir Chowdhury ID: 211219136 The sizes of the digests were estimated by constructing a standard curve (Figure 3) for DNA using the 1Kb ladder. An agarose gel electrophoresis was also performed using the double digested PCR product (B. licheniformis DNA with amyE region amplified by PCR). Figure 4 illustrates this gel. Lane 5 contains the 1kb DNA ladder and lane 7 contains the double digested PCR product. Lane 7 shows two bands. The first band is 1.6 Kb in length and is the Nde1 and BamH1 double digested DNA. The second band is most likely due to the presence of primers present in the PCR product. The sizes of the DNA bands were estimated by constructing a standard curve for molecular weight (Figure 5) using the 1 Kb DNA ladder. The cleaved amyE gene and the cleaved plasmid were ligated and the resulting recombinant DNA was used to transform E. coli competent cells. The pAMY8 amylase positive plasmid was used for transformation as a positive control. The transformed cells are plated in LB + ampicillin agar plates illustrated in figure 6. A colony PCR reaction is performed using two colonies from the plate. The PCR products are then run on an agarose gel. Figure 7 illustrates the image of this gel. Lane 1 contains the 1Kb DNA ladder. Lanes 2 and 3 contain our colony PCR product. The 1 st band in lane 1 corresponds to the amyE gene with a length of 1.6 Kb. The second band is unrelated DNA. The Second Lane doesnt contain the band corresponding to the amyE gene, indicating that the colony used for this sample was not transformed. Figure 8 illustrates the image of another gel that contained the amylase positive plasmid pAMY8 in lane 8. The band with an estimated size of 1.6 Kb corresponds to the amyE gene.

Tabashir Chowdhury ID: 211219136

Figure 4 Agarose gel of B. licheniformis cDNA digest with NDE1 and BamH1 restriction endonucleases. Lane 1 and 5 contains the 1Kb ladders and lane 7 contains our Nde1 /BamH1 restriction digest of B. licheniformis cDNA. Lane 3 contains the same restriction enzyme digested DNA prepared by group 4.

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Tabashir Chowdhury ID: 211219136

Figure 5 The standard curve for the agarose gel containing the B. licheniformis cDNA restriction digest. The y axis shows the log of molecular weight of each band of the 1KB DNA ladder. The x- axis shows the absolute distance travelled in the gel by the bands in the 1 Kb ladder.

Table 2 Data from the gel containing the cDNA digests. The log of molecular weight in Kb for the DNA in the 1kb DNA ladder and the respective absolute migration distance by each DNA fragment in the ladder

Log of Molecular Weight Kb 4 3.903 3.7782 3.698 3.602 3.477 3.301 3.1761 3 2.699

Absolute distance (mm) 10 11 12 13 14.5 15 17 19 22 27

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Tabashir Chowdhury ID: 211219136

Figure 6 Agar plate containing LB + ampicillin. Transformed E coli colonies are pointed out by the arrows. Two colonies were picked from the plate and used for the colony PCR.

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Tabashir Chowdhury ID: 211219136

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Tabashir Chowdhury ID: 211219136

Figure 8 Agarose gel of Colony PCR performed on the transformed E.coli colonies. Lane 5 contains the 1KB DNA ladder and lane8 contains the amylase positive plasmid pAMY8, which is a positive control in this experiment. The highlighted band corresponds to the 1.6 Kb long amyE gene present in the plasmid. Lanes one and 4 are empty, and lanes 2, 3,6 and 7, contain the colony PCR samples of the other groups.

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Tabashir Chowdhury ID: 211219136

Discussion
This experiment was used to clone the alpha amylase encoded by the amyE gene of B. licheniformis in to E. coli. The amyE amylase along with other amylases produced by the mesophile B. licheniformis is known to be active at temperatures in excess of 750C (5). There has been many studies done regarding other alpha amylases from B. licheniformis, such as the amyN encoded alpha amylase (6), and the amyL (7) alpha amylases which have shown close to 92% sequence homology with the amyE alpha amylase (7). The cDNA from B. licheniformis was extracted using a phenol: choloroform extraction followed by ethanol precipitation. During organic extraction, protein contaminants are denatured and partition either with the organic phase or at the interface between organic and aqueous phases, while nucleic acids remain in the aqueous phase. Phenol used in this protocol is buffered to prevent oxidized products in the phenol from damaging the nucleic acid (8). Isoamyl alcohol is used to prevent excessive foaming during the extraction. The aim of the extraction was to obtain pure high molecular weight DNA from the B. licheniformis cells. There have been other studies that involved the single step DNA isolation used acid guanidinium thiocyanate-phenol-chloroform extraction and achieved remarkable results (9). Our extracted DNA had a molecular weight close to 8Kb (refer to figure1). UV spectroscopy of the DNA sample provided an absorbance ratio A260/A280 of 2.01, indicating a pure DNA sample. The typical A260/A280 for isolated DNA is 1.9. A smaller ratio indicates increased contamination by protein (8). PCR was used to amplify a region of the amyE gene in the B. licheniformis cDNA. The forward primer was located at nucleotides 700-717 of the B. licheniformis DNA sequence, the reverse primer is located at nucleotides 1112 -1132 of the DNA sequence.

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Tabashir Chowdhury ID: 211219136 These two primers amplified PCR product of 433bp that includes the ROA and the two primer sequences. The amplified PCR product was cleaned up according to the Qiagen PCR reaction clean up protocol to purify the fragments from primers, nucleotides, polymerases, and salts leaving the DNA sample ready for other reactions. The PCR product is digested with two different restriction enzymes, Nde1 and BamH1. These enzymes cleave each DNA strand at a precise distance from the 5 end of the recognition sequence producing a restriction fragment with overhanging single stranded ends 1.6 kb in length(4) (refer to figure 4 and 5) . The pET-15b plasmid DNA is also cleaved with the same restriction enzymes and produces a fragment with complementary sticky ends, of 1.6 Kb in length (refer to figure 2 and 3). This facilitates the annealing of the plasmid and the cleaved DNA in the presence of T4 DNA ligase. The result was a recombinant pET-15b plasmid with the amyE gene fragment of B. licheniformis. This recombinant plasmid DNA is used to transform E. coli cells that are made competent by treatment with ice cold solutions of divalent cations (4) this weakens the cell wall and allows the E. coli cells to take up the recombinant plasmid DNA, this is further facilitated by subjecting the mixture to heat shock treatment which involves exposing the cells to a brief pulse of heat. The transformed E. coli cells are selected via the antibiotic resistance gene Ap (4643-5500) encoded by the pET-15b plasmid, which allows the E. coli to grow in the presence of ampicillin (4), since only the transformed E. coli cells can survive; it provides an ideal way to identify transformed cells. Two colonies of transformed bacteria were used to perform a PCR. Figure 7 shows the agarose gel electrophoresis image of the PCR product. The band in lane one is 1.6 Kb in length. According to a study done by Sahm et al. 1996, the amyE gene from T. thermosulfurigenes EM1 was determined to be

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Tabashir Chowdhury ID: 211219136 1.3Kb in length coding for a protein of 447 amino acids in length, with a molecular weight of 48,899 Da (10). The amyE gene cloned in our experiment has a higher size then the T.thermosulfurigenes amyE gene, however both genes are similar in length, which indicates homology between the two genes. This provides evidence to support our conclusion that the amyE gene was successfully cloned into the E. coli.

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Tabashir Chowdhury ID: 211219136

Questions
1. Cells are broken open by both physical (glass beads) and chemical (organic solvents) means. The organic solvents also denature nucleases (and other proteins) as soon as the cells are lysed. 2. DNA can be separated from other cellular components by extraction with phenol and chloroform, which denature and precipitate proteins, dissolve lipids, and remove some polysaccharides. 3.
Both phenol and chloroform are toxic compounds; working in a fume hood minimizes contact with the vapors.

4.

When a solution of DNA is mixed with 0.1 volume of 3 M sodium acetate, pH 5.2, and 2-2.5 volumes of 95% ethanol, the DNA precipitates out of solution.

5.

Ethidium bromide is a flat planar molecule that slides between the stacked base pairs of DNA. This is called intercalation.

6.

If the electrodes were reversed, the DNA would run off the short end of the gel above the wells and into the buffer chamber Exercise 7.1

1.

Reading frame 1: Ser Asp Asp Leu Gln Pro * Thr Reading frame 2: Gln Met Thr Tyr Ser His Lys Arg Reading frame 3: Arg * Leu Thr Ala Ile Asn Val Reading frame 4: Leu His Ser Val Ala Met Phe Thr Reading frame 5: * Ile Val * Leu Trp Leu Arg Reading frame 6: Gln Ser Ser Lys Cys Gly Tyr Val

2. 3.

Reading Frames 1, 3, and 5 Reading Frames 2 and 4

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Tabashir Chowdhury ID: 211219136

Exercise 7.2

1. 2. 3. 4. 5.

Reading frame 1 181, 289 Met 181

TAG, 1717 512 amino acids. Molecular weight = 512x110 da = 56,320 da

References

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Tabashir Chowdhury ID: 211219136 1. Maureen Barlow Pugh, (2000). Stedman's Medical Dictionary (27th ed.). Baltimore, Maryland, USA: Lippincott Williams & Wilkins. p. 65.

2. Rothstein, David Devlin, Patricia and CATE, Richard. Expression of alpha-Amylase in Bacillus licheniformis. Biogen Research Corp., Cambridge, Massachusetts 02142.

3. McWethy, S.J. and Hartman, P. A. (March, 1977). Purification and some properties of an extracellular alpha-amylase from Bacteroides amylophilus. J Bacteriol. 1977 March; 129(3): 15371544. 4. Thiel, T., Bissen, S., and Lyons, E.M. (2002) Biotechnology: DNA to Protein A Laboratory Project in Molecular Biology. McGraw-Hill, New York, pp.45-60. 5. Ingle, M. B., and R. J. Erickson. 1978. Bacterial a-amylases. Adv. Appi. Microbiol. 24:257-278.

6. Hmidet, N. et al. (May, 2008). Purification and biochemical characterization of a novel -amylase from Bacillus licheniformis NH1: Cloning, nucleotide sequence and expression of amyN gene in Escherichia coli. Process Biochemistry, Volume 43, Issue 5, Pages 499-510.

7. Gray, G. et al. (May 1986). Structural Genes Encoding the Thermophilic oLAmylases of Bacillus stearothermophilus andBacillus licheniformis. Journal of Microbiology, May 1986, p. 635-643, Vol. 166 No. 2 0021-9193/86/050635-09. Genencor, Inc., South San Francisco, California 94080, and Department ofBiochemistry, Cornell University, Ithaca, New York148312. 8. The Enzyme Handbook, Vol. 4, Schomburg, D., and Salzmann, M., Springer-Verlag (Berlin Heidelberg: 1991), EC 3.2.1.1, p. 7.

9. Chomczynski, P., Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical Biochemistry,

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Tabashir Chowdhury ID: 211219136 Volume 162, Issue 1, April 1987, Pages 156-159, ISSN 0003-2697, 10.1016/00032697(87)90021-2.

10. Sahm, K. et al. (Feb. 1996). Molecular Analysis of the amy Gene Locus of Thermoanaerobacterium thermosulfurigenes EM1 Encoding Starch-Degrading Enzymes and a Binding Protein-Dependent Maltose Transport System. JOURNAL OF BACTERIOLOGY, Feb. 1996, p. 10391046, Vol. 178, No. 4 0021-9193/96. Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS, United Kingdom.

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