Results

Discuss about the Bacillus Subtilis.

Bacillus subtilis is a Gram-positive non-pathogenic bacillus involved in the formation of heat resistant and dormant spores (Logan, Niall and Paul). It is the most characterized microorganisms among the various Gram-positive bacteria present. It genetic material can easily be subjected to manipulation and as a result widely used in genetic engineering. It is used commercially for the production of a variety of enzymes, vitamins, flavoring agents and in the production of industrial nucleotides (Capozzi et al.).

Genomic DNA libraries carry the entire genomic DNA sequence of the organism. The genomic DNA library can be used to determine the whole genome sequence of an organism helps to determine the phenotypes regulated by the genes, determination of mutations present in the genome and in the production of proteins expressed from the respective genes for commercial use.

Genetic engineering requires the use of recombinant DNA technology in order to carry out genetic manipulation of an organism. Genetic engineering can also be used to generate mutations of genes, whose functions are not known (Gaj, Gersbach, and Barbas). Various types of products like insulin, industrial enzymes and even the human growth hormone has been produced by the use of genetically modified organisms (GMOs).  Genetically modified crops can also be produced using this technique (Bawa and Anilakumar). The overall purpose of this report is “to generate the genomic DNA library of B. subtilis and carrying out the necessary steps to determine its efficacy”.

Lab 3 results

The concentration of the B. subtilis genomic DNA isolated in Lab 2 was 44ng/µl. EcoRI and HindIII restriction enzymes were used to digest the genomic DNA. The digested fragments were then ligated into the EcoRI and HindIII digested empty vector pUC18 to obtain recombinant plasmids carrying the various genetic regions of the B. subtilis genome. The recombinant plasmids thus obtained were introduced into the Escherichia coli DH5α cells. Restriction digestion of the genomic and plasmid DNA was carried out at  37ºC for 1 hour, then incubation at 80 ºC for 10 minutes was carried out to inactivate the restriction enzymes. The digestion products were at first heat inactivated at 45 ºC for 5 minutes to remove the reannealed digested products. This was followed by the actual ligation step at 18 ºC for 30 minutes. The ligation products were incubated at 65 ºC for 10 minutes to inactivate the ligase. The ligated products were transformed into E. coli DH5α cells plated on X-gal, IPTG and Ampicillin containing LB agar plates.

Discussion

Lab 4 results

The ratio of the blue to white colonies were 3:8. The control plates used were the digested and re-ligated pUC18 plates, which gave rise to blue colonies due to the absence of an insert. The non-transformed and no T4 DNA ligase plates did not show the presence of colonies, while an already prepared recombinant plasmid was used as the positive control, which gave rise to white colonies (Apppendix, Figures 1-4). The recombinant clones obtained in the experimental plates were then used to isolate the recombinant plasmid. A white recombinant colony was selected.  The concentration of the isolated plasmid DNA was 621.1ng/µl and the 260:280 ratio was 2.12. The isolated recombinant plasmid was single digested with HindIII and double digested with EcoRI/HindIII. This helped to confirm the presence of the insert.

 Lab 5 results

The distances travelled by the bands in the DNA ladder were calculated (Appendix, Table 1, Figure 5) and plotted with respect to the length of the DNA bands in base pairs. The X- axis of the standard curves generated represents the DNA length in base pairs and the Y- axis represents the distance travelled in mm. A logarithmic trendline was generated in case of both the standard curves (Appendix, Figures 6 and 7). Calculations using the equations in the standard curves  were done to determine the sizes of the DNA bands in the gel (Appendix, Figure 5). The sizes of the DNA bands are provided in Table 2 (Appendix).

In gel 1, the pUC 18 plasmid had three bands of sizes 12088, 5115 and 4023bp, respectively. The double digested pUC18 and the genomic DNA lanes showed one band of sizes approximately 2697bp and 13359bp, respectively. The control uncut recombinant plasmid had 4 bands of sizes 12088, 5115, 4023 and 2440.6bp. The single digested plasmid had one band of size 4769bp. The double digested control plasmid produces 2 bands of sizes 3165 and 1353bp, respectively (Appendix, Figure 5). In gel 3, the genomic DNA had one band of size 11849bp. The uncut plasmid had 2 bands of sizes 3605 and 2208bp. The single digested plasmid had one band of size 3605bp. The double digested product had 2 bands of sizes 2697 and 916bp. The foreign DNA insert was approximately 916bp (Appendix, Figure 5). While the single digested product was 3605bp approximately, the double digested product adds up to 3613bp. The single and double digestion of the control recombinant plasmid yielded one and two bands, respectively (Appendix, Figure 5).

Additional experiments

This report describes the preparation of a genomic DNA library. Digestion and ligation of the genomic DNA inserts into the vector pUC18 yielded the recombinant clones. The number of white colonies were more than the number of blue colonies. The recombinant plasmid containing colonies were white because the lacZ gene present in the multiple cloning site (MCS) of the vector gets disrupted due to the addition of the insert (Davis). The blue colonies carried the empty vectors, which were obtained due to their undergoing only single digestion. As a result, they were able to break down the X-gal substrate giving rise to the blue color. This is because the lacZ gene remained intact producing functional beta galactosidase. Moreover, the double digestion of the recombinant plasmid yielded DNA bands of sizes 2697 and 916bp, which are the vector and insert bands, respectively. Additionally, the single digested product gave a single band of size 3605bp.

The problems that were encountered were the absence of colonies. Absence of colonies can be due to improper plasmid and genomic DNA purification, which can hamper the digestion step (Surzycki). Star activity can result in non-specific digestion of the genomic and plasmid DNAs (Pingoud, Wilson and Wende). Moreover, restriction enzyme inactivation is crucial otherwise it can interfere with the ligation steps. Lastly, proper generation of competent cells are necessary for successful transformation (Tu et al.). Other interesting results that were obtained is the presence of white colonies in the pUC 18 plates and blue colonies in the experimental plate. The blue colonies in the experimental plate indicates the inappropriate digestion of any one of the enzymes, thereby resulting in re-ligation of the vector. The control plate containing pUC18 is expected to produce blue colonies, however, mutations in the lacZ gene can case the appearance of white colonies.

Additional experiments includes polymerase chain and cycle sequencing to confirm the presence of the insert in the desired vector (Hoseini and Sauer).

The genomic DNA library can be used identify genes that express commercially valuable protein products. Genomic DNA libraries can also be used to identify genetic alterations of an organism and also identify the genetic regulators that modulate an organism’s genetic circuitry.

The purpose is to overexpress the desired gene and produce large quantities of proteins, which will be subjected to further purification.

The blue white screening is based on the theory of α-complementation. lacZ encodes the enzyme β-galactosidase, which is a tetramer having 2 α and 2 ω fragments. E. coli cells that lack the α fragment, produce non-functional β-galactosidase. However, the α fragment can be introduced by the introduction of a plasmid expressing the α fragment in trans. The lacZ gene present in the MCS gets disrupted by the addition of an insert (Blau and Wehrman). IPTG and X-gal is added to the LB media. IPTG acts as the gratuitous inducer and X-gal functions as the chromogenic substrate. Non-functional LacZ cannot degrade the substrate and produce white coloration, while functional LacZ degrades the substrate to produce blue coloration. The recombinant plasmids are identified by the white coloration of the colonies (Chaudhuri). DH5α cells are ΔM15 strains, where 11-41 amino acid residues (α fragment) from the N-terminal of LacZ is deleted and subsequently the residual ω fragment is inactive. Thus, the DH5α strain is suitable for such a screening as blue colonies will appear only when a plasmid expressing the α fragment is introduced (Aguilera and Aguilera-Gómez).

Reference List

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Bawa, A. S., and K. R. Anilakumar. “Genetically modified foods: safety, risks and public concerns—a review.” Journal of food science and technology 50.6 (2013): 1035-1046.

Blau, Helen M., and Thomas S. Wehrman. “Detection of protein translocation by beta-galactosidase reporter fragment complementation.” U.S. Patent No. 8,586,294. 19 Nov. 2013.

Capozzi, Vittorio, et al. “Lactic acid bacteria producing B-group vitamins: a great potential for functional cereals products.” Applied microbiology and biotechnology 96.6 (2012): 1383-1394.

Chaudhuri, Keya. Recombinant DNA Technology. The Energy and Resources Institute (TERI), 2013.

Davis, Leonard. Basic methods in molecular biology. Elsevier, 2012.

Gaj, Thomas, Charles A. Gersbach, and Carlos F. Barbas. “ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering.” Trends in biotechnology 31.7 (2013): 397-405.

Hoseini, Sayed Shahabuddin, and Martin G. Sauer. “Molecular cloning using polymerase chain reaction, an educational guide for cellular engineering.” Journal of biological engineering 9.1 (2015): 2.

Logan, Niall A., and Paul De Vos. “Bacillus.” Bergey’s Manual of Systematics of Archaea and Bacteria (2015).

Pingoud, Alfred, Geoffrey G. Wilson, and Wolfgang Wende. “Type II restriction endonucleases—a historical perspective and more.” Nucleic acids research 42.12 (2014): 7489-7527.

Surzycki, Stefan. Basic techniques in molecular biology. Springer Science & Business Media, 2012.

Tu, Qiang, et al. “Room temperature electrocompetent bacterial cells improve DNA transformation and recombineering efficiency.” Scientific reports 6 (2016)