PCR Amplification Of Alpha-Amylase Gene In Bacillus Subtilis

Iodine test for starch

Discuss about the A-amylase Gene using Polymerase Chain Reaction.

Bacillus subtilis is a Gram-positive, rod-shaped and flagellated bacterium that are natural inhabitants of soil and vegetation as well as our gastrointestinal tract. It has a circular chromosome like most of the prokaryotes with approximately 4214.8 Kb (Kunst et al., 1997) coding for more than 4000 proteins (Kobayashi et al., 2003). Out of the 4000 proteins many of them functions in metabolic activities which includes enzymes that are involved in breaking down carbon sources. One of the enzymes is the α-amylase which is required for breakdown of carbohydrates into monomers (glucose and maltose) (Yamazaki et al., 1983). The enzyme is produced by the bacterium and is secreted to the external environment to breakdown starch in the immediate vicinity of the organism. After breaking down complex carbohydrate sources the simplified form such as glucose is taken up by the cell for utilization by the cell into its cytoplasm. 

The ability to secrete α-amylase can be tested by culturing the bacterium on agar medium supplemented with starch and checking by iodine. Iodine has the ability to detect starch by changing colour from brown to purple. In case of absence of starch there will be no colour reaction and iodine will not change colour (Ortlepp, Ollington, & McConnell, 1983). This means that B. subtilis which produces the α-amylase enzyme will digest starch into smaller constituents and iodine will not change colour. By using organism that cannot produce the enzyme and comparing with B. subtilis we can determine the difference in iodine reaction of the two organisms on an agar plate supplemented with starch.

After determination of the presence of the enzyme a procedure of whole genome isolation from the organism with an aim to purify the coding gene for α-amylase will be carried out. The genomic isolation follows a procedure of destructing the bacterial cell wall and precipitating the genomic DNA. Lysis is performed using an anionic detergent, SDS, and lysozyme along with proteinase K. SDS destroys the lipid bilayer and precipitates protein, whereas lysozyme and proteinase K cleaves protein into amino acids leading to destruction of cell wall and membrane of the bacterium. Further, addition of RNase will destroy all the ribonuclease present in the lysate. The constituent protein is then precipitated with 1:1 phenol: chloroform mixture. The solution that remained after organic phase extraction contains the DNA which is purified by using silica resins as stationary phase. Silica groups are negatively charged and will bind with Na+ ions present in the mobile phase and will form a net positive charge. The positive charge will then attract DNA which is negatively charged which is then washed with ethanol and eluted using an elution solution. The whole process can be carried out in a specially designed spin column that are commercially available. The isolated DNA is then quantitated and quality checked by spectrophotometer reading at 260 and 280nm. 

Genomic DNA isolation and PCR amplification

The isolated DNA can then be used as a template for PCR amplification of the α-amylase gene using gene specific primers. The amplified product can then be checked using agarose gel electrophoresis which separates DNA according to its size. As DNA is negatively charged it will travel towards positive charged electrode and the movement in an agarose medium is restricted by size allowing smaller molecules to travel ahead of larger ones. By using a standard marker we can determine the size of the amplified product.

Iodine test for starch: The iodine test for presence of starch indicated a negative result on the side of the agar plate growing B. subtilis with no colour change of iodine, and a positive reaction for E. coli culture, iodine turned purple [Figure 1]. The result indicates absence of starch on the side of the plate with B subtilis and presence of starch on E. coli culture.

Genomic DNA isolation and PCR amplification: The genomic DNA which was isolated from a culture of B. subtilis was checked with spectrophotometer for its purity and concentration. The absorbance at 260 nm UV was 0.25 and at 280 nm was 0.133. A 260/280 reading of 1.87 and a concentration of 12.5 µg/ml. The concentration was estimated using the formula: A260 × concentration × 50 µg/ml. In a reaction volume of 20 µL PCR reaction, 8µL of the isolated genomic DNA was used to get a final concentration of 0.1 µg in 20 µL [Table 1].

Determination of size of PCR product: The amplified product of PCR was electrophoresed on an agarose gel along with a standard DNA 100bp ladder. The gel was photographed [Figure 2] and distance travelled by each standard band was measured [Table 2] to establish a standard curve of distance migrated on Y axis and log of base pairs of standard on X axis. A linear trendline equation was established with R² value of 0.99 which is the best fit [Figure 3]. Using the equation of the chart the size of the amplified band was estimated as 725 base pairs. We could not observe any product at the negative control well.

subtilis houses a gene for α-amylase and produces the enzyme for utilization of starch into its external environment and utilizes the product for its energy needs. The degradation of starch is evident from the iodine test which does not show a colour change from brown to purple in the B. subtilis culture. The result that we obtained is similar to previous experiments by other researchers (Swain & Ray, 2007). After confirmation of presence of the enzyme by the iodine test we isolated the genomic DNA from the organism which was used for PCR reaction to amplify the gene. We determined the quality of the isolated DNA by measuring absorbance at 260 nm and 280 nm UV light which yielded a A260/A280 of 1.8 indicating a pure DNA sample.

Determination of size of PCR product

In order to amplify the α-amylase gene we used a PCR reaction. PCR is a dynamic method that specifically amplifies target DNA sequence by using specified forward and reverse primers to limit the reaction. The primer binds to the target DNA sequence in a Watson-crick base pairing method and serves as a 3’OH free end for addition of nucleotides complementary to the target sequence by the activity of Taq polymerase (a thermostable DNA polymerase). For this dNTPs are provided in abundance for the reaction to proceed. PCR is carried out in three steps which is repeated over around 30 times leading to exponential multiplication of the product; Denaturation which separates the double stranded DNA, annealing in which primers anneal to targets and extension in which DNA strands are made by Taq polymerase. PCR can be used to amplify any DNA sequence provided that specific primers for the target are used and the annealing temperature is optimized for amplification. Sometimes when an mRNA is used for amplification of the target gene it should be first converted to cDNA by reverse transcriptase in a process called reverse transcription. The product of the amplification is then checked by agarose gel electrophoresis.

Agarose gel electrophoresis is a method in which DNA can be separated based on size. Agarose is a gelling agent and forms a gel-like substance, the hardness of which is dependent on the percentage of agarose. The higher the percentage of agarose smaller is the pore size and hence retardation in mobility of the DNA sample therefore a higher gel percentage would reduce distance travelled. When electrophoresed in buffer (ion carrier such as TAE or TBE) the DNA moves from negative to positive electrode with smaller fragments moving faster than bigger ones. In our experiment we determined the size of the amplicon using a standard curve plotted with distance migrated by standard DNA base pair. The size of the amplicon was determined to be 724 base pair which was the same size as positive control band. However, the size of the gene is approximately 1539 base pairs (Ortlepp et al., 1983; Yamazaki et al., 1983; Yang, Galizzi, & Henner, 1983), which means we amplified only a part of the gene. We successfully amplified the gene for α-amylase from B. subtilis.

References 

Kobayashi, K., Ehrlich, S. D., Albertini, A., Amati, G., Andersen, K., Arnaud, M., . . . Bessieres, P. (2003). Essential Bacillus subtilis genes. Proceedings of the National Academy of Sciences, 100(8), 4678-4683.

Kunst, F., Ogasawara, N., Moszer, I., Albertini, A. M., Alloni, G., Azevedo, V., . . . Danchin, A. (1997). The complete genome sequence of the gram-positive bacterium Bacillus subtilis. Nature, 390(6657), 249-256. doi: 10.1038/36786

Ortlepp, S. A., Ollington, J. F., & McConnell, D. J. (1983). Molecular cloning in Bacillus subtilis of a Bacillus licheniformis gene encoding a thermostable alpha amylase. Gene, 23(3), 267-276.

Swain, M., & Ray, R. (2007). Alpha?amylase production by Bacillus subtilis CM3 in solid state fermentation using cassava fibrous residue. Journal of Basic Microbiology, 47(5), 417-425.

Yamazaki, H., Ohmura, K., Nakayama, A., Takeichi, Y., Otozai, K., Yamasaki, M., . . . Yamane, K. (1983). Alpha-amylase genes (amyR2 and amyE+) from an alpha-amylase-hyperproducing Bacillus subtilis strain: molecular cloning and nucleotide sequences. Journal of bacteriology, 156(1), 327-337.

Yang, M., Galizzi, A., & Henner, D. (1983). Nucleotide sequence of the amylase gene from Bacillus subtilis. Nucleic acids research, 11(2), 237-250.