Creating A Genomic DNA Library Of Bacillus Subtilis Using Recombinant Plasmids

Bacillus subtilis as a microorganism

Question:

Discuss about the Genomic DNA Liabrary.

Bacillus subtilis is a Gram positive and rod shaped microorganism. It produces dormant and heat resistant spores and is non-pathogenic (Leggett et al., 2012). The genomic DNA of B. subtilis is circular and is approximately 42,14,630 base pairs, with a GC content of 43.5% and encodes 4100 proteins (van Dijl & Hecker, 2013). It is a soil bacterium and is used for the production of many industrial products like commercial enzymes like proteases and amylases, vitamins like riboflavin, supplements like poly gamma glutamic acid, flavoring agent ribose, industrial nucleotides, among others (Singh et al., 2016). It’s genome can be easily manipulated and as a result can be used in genetic engineering. It is the best characterized of among all the Gram-positive bacteria species having low GC content.

Genomic DNA libraries are collections of genomic DNA sequences from an organism. This type of library consists of all the gene sequences present in the genome of the organism. Each clone consists of at least on one copy of the DNA sequences or genes present in the genome. The whole genome of the organism is represented by a set of genes or DNA segments inserted into a vector DNA molecule. Genomic library serves many purposes. It helps to determine the whole genome sequence of an organism, helps in the study of the functions of the genes or regulatory sequences; it helps to determine the presence of any genetic alterations or mutations, particularly in cancer tissues, helps in expression of genes that encodes proteins of industrial or commercial importance. It can also help in the production of novel pharmaceutical products (Rohland & Reich, 2012).

Genetic engineering is the direct manipulation of the genetic organization of an organism using the technique of recombinant DNA technology. It helps in the transfer of genes from one organism and its subsequent expression in another organism. Apart from inserting new genes, genetic engineering also involves the mutation of knocking out of genes in an organism. Genetically modified organisms, particularly bacteria have been used in the past to produce insulin, human growth hormone, industrial enzymes that are used in laundry detergents, among others. This technique has also been used for the production of genetically modified crops or GMOs (Nielsen, 2013). The overall purpose of this report is “to create a genomic DNA library using the genome of the organism B. subtilis, followed by its subsequent confirmation steps to determine success of the process”.

Genomic DNA Libraries

Lab 3 results

The genomic DNA of B. subtilis that was isolated during Lab 2 were used for the subsequent cloning steps. The concentration of the genomic DNA was 44ng/µl. The isolated genomic DNA was used to create the recombinant plasmids present in the genomic DNA library. The genomic DNA was digested with the restriction enzymes EcoRI and HindIII to yield the different DNA fragments or inserts that will be ligated to the similarly digested plasmid DNA vector (pUC18) to create recombinant plasmids that are transformed into the Escherichia coli DH5α cells. The samples subjected to restriction digestion were incubated at 37ºC for 1 hour, followed by incubation at 80 ºC for 10 minutes to inactivate the restriction enzymes. The respective digestion products of the genomic DNA and plasmid DNA were subjected to ligation. For this, the sample was incubated at 45 ºC to denature the reannealed digested products, followed by ligation at 18 ºC for 30 minutes. The ligation reaction was then incubated at 65 ºC for 10 minutes. The ligated products were subsequently transformed into E. coli DH5α cells. The transformants were plated on LB agar plates containing X-gal, IPTG and Ampicillin.

Lab 4 results

The ratio of blue to white colonies obtained after incubation of the transformed plates were 3:8. The control plates that were used includes positive control plates, where an previously prepared recombinant plasmid was transformed into the E. coli cells, which gave rise to white colonies indicating the presence of recombinant plasmids. The no T4 ligase control plate did not show the presence of any colonies, the no transformation plate also did not show the presence of the colonies and the digested and re-ligated pUC18 plasmid DNA gave rise to blue colonies (Apppendix, Figures 1-4). The recombinant plasmid DNA was isolated from one of the white colonies obtained from the experimental plates. The concentration of the plasmid DNA was 621.1ng/µl and the 260:280 ratio was 2.12 as determined by sing the Nanodrop Spectrophotometer. The isolated recombinant plasmid was subjected to single and double restriction digestion by the use of HindIII and EcoRI/HindIII, respectively. This was used to confirm the presence of the insert in the plasmid DNA. Single digestion of the recombinant plasmid will produce a shift with the digested vector DNA pUC18, while double digestion will help to obtain the insert present in the recombinant plasmid. The digested products were run in an agarose gel with respective controls and DNA ladder in order to determine the size of the insert that was ligated into the pUC18 plasmid DNA.

Genetic engineering

Lab 5 results

The distances (in mm) travelled by the DNA bands present in the DNA ladder were determined for both the gels 1 and 3(Appendix, Table 1, Figures 5 and 6) and subsequently plotted along with the length of the DNA bands in base pairs. The X- axis represents the DNA length and the Y- axis represents the distance travelled. A logarithmic trendline was found to be appropriate for the DNA ladders of both Gel 1 and 3 (Appendix, Figures 7 and 8). Calculations were carried out using the equation present in the standard curves to determine the sizes of the DNA bands present in the gel 1 (control samples, Figure 5) and gel 3 (Experimental gel, Figure 6). The sizes of the DNA bands are provided in Table 2 (Appendix).

In gel 1, the uncut pUC 18 had three bands of sizes 8103, 3827 and 2980bp, respectively. The double digested pUC18 and the control genomic DNA had one band of sizes approximately 2697bp and 12088bp, respectively. The control uncut, single digested and double digested recombinant plasmid had two bands each of sizes approximately 4447.1 and 2208.3bp, 2697 and 735bp, 2697 and 665.1bp, respectively (Appendix, Figure 5). In gel 3, the genomic DNA, uncut and single digested recombinant DNA had only one band of sizes approximately 13359bp, 3640bp and 3400bp, respectively. The double digested recombinant DNA had two bands of sizes approximately 2697bp and 692bp, respectively. The foreign DNA insert obtained after double digestion of the recombinant plasmid with EcoRI/HindIII was approximately 692bp (Appendix, Figure 6). While the single digested product was 3400bp approximately, the sizes of the DNA bands in the double digested product adds up to 3389bp, which is more or less the same as the DNA size obtained in the case of single digestion. The single digestion of the control recombinant plasmid yielded two bands, while the single digestion of the experimental recombinant plasmid yielded one band (Appendix, Figures 5 and 6).

The aim of this report was to generate a genomic DNA library. For this, the genomic DNA of B. subtilis and pUC18 plasmid DNA were digested and ligated. Transformants obtained showed that the ratio of the blue to white colonies were 3:8. Thus, the number of clones containing recombinant plasmids were much higher than the clones carrying the empty vector pUC18. Therefore, maximum number of inserts obtained after digestion of the genomic DNA had undergone ligation with the similarly digested plasmid DNA supporting the success of the experiment. The white colonies were obtained as a result of disruption of the lacZ gene present in the multiple cloning site (MCS) of the vector. However, some single digested vectors were present that gave rise to the blue colonies, since the lacZ gene remained intact and utilized the X-gal substrate to produce the blue color. Moreover, the recombinant plasmid obtained was confirmed by double digestion, which yielded DNA bands of sizes 2697 and 692 bp. These two are the sizes of the vector pUC18 (2697bp) and the foreign DNA insert (692bp). Moreover, the single digested product gave a single DNA band of size 3400bp, which is the same when the sizes of the double digested products are added up. The sizes of the DNA bands obtained after digestion of the control recombinant plasmids indicated that the parent plasmid here was also pUC18. Moreover, the single digestion of this control recombinant plasmid revealed that the enzyme used had two sites, thereby giving rise to two bands, like that in the case of double digestion, however the sizes of the inserts obtained in both the cases were slightly different.

Experimental Process and Results

The problems that were encountered in the process were no colonies were obtained in the experimental plate. This result could be due to the presence of various discrepancies. These include: (1) improper purification during genomic and plasmid DNA preparation. Presence of ethanol, phenol and chloroform can inhibit or interfere with restriction digestion (Naushad et al., 2012). (2) Improper inactivation of restriction enzymes can interfere with the subsequent ligation steps, preventing successful ligation. (3) Inappropriate preparation of competent cells can also prevent the uptake of the recombinant DNA, thereby preventing the appearance of colonies on the plates (O'Connell, 2012). (4) Star activity can cause the DNA could be cut at non-specific sites by the restriction enzymes (Lundin et al., 2015). Other odd results that were obtained is the presence of blue colonies in the experimental plate and the presence of white colonies in the pUC 18 control plates. The blue colonies in the experimental plate indicates the presence of single digested or undigested plasmid vectors that have re-ligated, even after incubation at 45ºC to denature the reannealed products. This may be due to inappropriate or insufficient incubation at the desired temperature, since, only white colonies are expected in the experimental plate. The control plate containing only pUC18 is expected to give rise to only blue colonies, however, mutations in the lacZ gene present in the plasmid can give rise to white colonies. Another odd result was that when plasmid DNA was isolated from some of the white colonies from the experimental plates and subjected to double digestion did not give rise to the insert DNA, indicating that those white colonies consisted of only pUC18, where the lacZ gene got mutated.

Additional experiments that can be carried out includes polymerase chain reaction using vector specific primers to amplify the gene of interest and subsequent sequencing to determine the DNA sequence of the insert (Erlich, 2015).

The genomic DNA library can be used to identify specific genes from the B. subtilis genome that can encode protein products of commercial value. After identification, large amounts of the desired protein product can be obtained by the addition of IPTG, which acts as a gratuitous inducer. Moreover, the insert can be cloned in the pET vectors and transformed into BL21(DE3) for production of large quantities of protein products.

The purpose of this application is the overexpression of the gene of interest to produce large quantities of protein products, which can be isolated and purified.

Conclusion and Issues Encountered

The blue white screening is based on the principle of α-complementation. It is based on the presence of the lacZ gene. lacZ encodes β-galactosidase, which is a tetramer having α and ω fragments. E. coli cells that lack the α fragment, have non-functional ω fragments and as a result the β-galactosidase is inactive. However, the functionality of the α fragment can be restored by the introduction of a plasmid expressing the α fragment in trans. Thus, the α and ω fragment gives rise to the functional β-galactosidase enzyme. The lacZ gene of the plasmid is present in the MCS and when an insert gets introduced into the MCS, the lacZ gene gets disrupted (Dooda et al., 2015). IPTG and X-gal is added to the LB media. IPTG acts as the inducer and X-gal acts as the chromogenic substrate. Non-functional lacZ gene product cannot degrade the substrate to produce blue color and instead gives rise to white colonies, while functional lacZ gene product degrades the substrate to produce blue colonies. The recombinant plasmids express non-functional LacZ and produces white colonies, while the empty plasmids express functional LacZ and produce blue colonies (Stevenson et al., 2013). DH5α cells are ΔM15 strains, in which N-terminal 11-41 amino acid residues (α fragment) of LacZ is deleted and the residual ω fragment alone is inactive. Thus, the DH5α strain is suitable for blue white screening as it will give rise to blue colonies only if a plasmid expressing the α fragment is transformed into the cells (Mahmoud et al., 2015).

Reference List

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