Bioinformatics of Bt Cry Toxins - Lab Report Example

Lab Report: Bioinformatics of Bt Cry toxins Lab Report: Bioinformatics of Bt Cry toxins Introduction The primary aim of this practical experiment is to demonstrate various ways of retrieving sequence information from cry genes as well as investigate the differences between natural bacterial and synthetic cry genes and the interrelatedness between various cry proteins. This is particularly important in enhancing the understanding of the changes that occur in Bacillus thuringiensis (Bt) Cry toxins in order for their expression to be optimized in transgenic plants. The insecticidal protein crystals have genes coding associated with large molecular mass and plasmid (Carlson 2010, p.65). Most of the cry protein genes formation is through cloning, sequencing and finally named cyt genes or cry genes. Over a hundred cry gene sequences is organized into thirty-two groups and other different subgroups based on their range of specificity and nucleotide similarities (Neale 2008, p.32). An example is the protein toxic used on lepidopteron insect, which belongs to the Cry 1, Cry 2, and Cry 9 groups (Bergeron 2013, 12). The toxins used against coleopteran insects include Cry 8, Cry 3, and Cry 7 and Cry 11a1 a subclass of Cry1 protein. There are several examples of nematocidal cry proteins, which include Cry 13, Cry 5, Cry 12 and Cry 14. Cry 2Aal a subgroup of Cry 2 protein, Cry 10, Cry 16, Cry 4, Cry 19, Cyt proteins and Cry 11 are poisonous to dipterans insects. B.thuringiensis strain carries more than one crystal toxin protein and, therefore, different strains of organisms can synthesize more than one crystal protein (Fisher 2009, 23). Transfer of plasmids in B.thuringiensis is the main agent of creating change in toxin genes. The synthesis of insecticidal crystal protein in bacteria takes place during its stationary phase of its life cycle growth. The proteins are stored in the mother cell. B.thurigienisis sporulated cells contain twenty-five percent of the dry weight of the protein. The soaring intensity of crystal protein formation in B.thuringiensis is controlled during different processes (Caballero, 2008, p.32). The processes are likely to occur at the post-translational, post-transcriptional and transcriptional levels. Specific sporulation genes control the expression of cry genes. Some of the cry gene expresses their action during vegetative growth. The expressions of cry gene are divided into sporulation independent and sporulation-dependent. Cry 1Aa gene is an exemplar of sporulation-dependent cry genetic material. It produces toxins against Lepidoptera (Jarrett, 2007, p. 56). This gene is depicted on the sporulation phase. Cry 3Aa gene is expressed during the stationary stage and vegetative growth. Its expression is more active in the vegetative phase than in the stationary phase. Stable cry m RNA contributes to increased production of toxins at the post-transcriptional stage. Cry m RNA has a half-life of ten minutes greater an average bacteria m RNA (Sweet, 2012, p.34). Cry1Aa stem loop configuration or the alleged transcriptional terminator functions as a positive retro regulator. The combination of the DNA material containing this terminator with the three end of the heterogeneous gene raises the half-life two to three times on their transcript. Three stem loop structures increases cry m RNA stability. An example is cry 1Aa transcriptional terminator, which increases the stability of cry m RNA by protecting it from degradation of exonucleotytic at the three ends (Hemming, 2011, 14). The mother cell compartment contains the proteins crystal in the state of crystalline inclusion the shape of the crystal depends on the composition of the protoxin (Jackson, 2012, p.45). The capacity of the protoxins to form crystals may reduce their activeness to immature proteolytic degradation (Leroy, 2012, p.15). The energy in the disulphide bonds, secondary structure of the protoxins and the presence of another B.thuringiensis affect the solubility and structure of the cry proteins. Materials and Methods The materials and bioinformatic tools used in the laboratory experiment included protein databases, DNA databases as well as specialized bioinformatic databases such as prosite and Worm base. In addition, a number of computer programs belong to the BLAST (Basic Local Alignment Search Tool) family of programs were also used to conduct homology searches on the databases to help locate sequences with similar structures and provide important clues about the potential functions of their genes or proteins. Although there are a number of BLAST servers available on the net, NCBI Blast server were preferentially used for the duration of the practical. The procedure included five simple protocols namely; retrieving a sequence from a given database, aligning two sequences to determine their similarities or differences, translating the sequence, locating similar sequences and finally aligning multiple sequences in order to help understand their interrelatedness. The first protocol/procedure involved retrieval of sequences from the NCBI database. The URL http://www.ncbi.nlm.nih.gov/ was typed on the web browser and from the results, nucleotide database was selected from the search drop down menu. Next, the accession DQ241675.1 was typed into the provided on the database search field and the search button was then pressed. After retrieving the results of the accession were displayed, its coding sequence link was clicked and FASTA format was selected in order to help find the regions of similarity between the sequences. The header and sequence displayed on the screen was then highlighted and copied to Notepad before being saved on a USB flash disk. Finally, the entire process was repeated for accession AY376665.1. In the second protocol, the URL http://www.ebi.ac.uk/muscle was typed in the address line of the web browser and the CDS sequence for accession DQ241675.1 and that of AY376665.1 both obtained from protocol 1 were then pasted into the provided box and submitted in order to align their sequences. The resulting alignment output was then copied, pasted and saved as “cds alignment” using the Noetepad. Protocol 3 entailed carrying out sequence translation using a bioinformatic translation tool known as Transeq. The Transeq program was launched by typing the URL www.ebi.ac.uk/emboss/transeq into the web browser address line. Next, the CDS for AY376665.1 was copied and pasted into the sequence field and the output was saved using Notepad and the file was named AY376665.1 aa. The whole process was repeated for DQ241675.1. In the forth protocol, homology search was conducted using BLAST program. Protein BLAST (blastp) option was selected on the NCBI server and the protein sequence of each accession was then pasted in before clicking BLAST. The final procedure (protocol 5) involved aligning multiple sequences in order to help understand their interrelatedness. The FASTA of the cds for accession DQ241675.1 (obtained from protocol 1), M89794, Y09787.1 and AY960853.1were pasted on the sequence alignment each at a time. Lastly, the results of the program were then run to provide alignment sequences illustrating the interrelatedness of the 3 sequences. Results and Discussion In sequence retrieval, genes are often named given accession numbers for easier identification and to retrieval of their DNA sequence from the databases. For example, the accession numbers accession DQ241675.1 and AY376665.1 were used to such the NCBI’s nucleotide database. The accession coding sequences for DQ241675.1 and AY376665.1 had a number of significant differences. One of the major differences between accession DQ241675.1 and AY376665.1 was the unequal lengths of the two coding sequences obtained from the results. On the other hand, significant differences between the two genes were also noted in terms of the observed sequence changes between the two CDS’s. According to many experts, these differences are particularly attributed to the fact that accession DQ241675.1 is a natural Cry1Ab toxin from Bacillus thuringiensis bacterium while accession AY960853.1 is obtained from a synthetic Cry1Ab gene. As a result, the observed differences between the two coding sequences are likely to be due to the changes that occurred on the Cry1Ab toxins in the process of producing a synthetic construct that can express itself in the transgenic plants (Jackson, 2012, p.34). Conclusion In summary, workshop practical have effectively revealed have a number of the differences between natural bacterial and synthetic cry genes as well as the interrelatedness between various cry proteins. Based on the results of the sequence retrieval and analysis, it can be safely concluded that Bacillus thuringiensis (Bt) Cry toxins usually undergo significant changes in order for their expression to be optimized in transgenic plants. References Bergeron, B. P. 2013. Case studies in genes and disease a primer for clinicians. Philadelphia, American College of Physicians. . Carlson, I. T. (2010). A study of genes affecting cyanogenetic glycoside content and other characters in Sudan grass, Sorghum sudanese (Piper) Stapf. Thesis (Ph. D.)--University of Wisconsin--Madison, 1955. Caballero, B., Allen, L. H., & Prentice, A. (2008). Encyclopedia of human nutrition. Amsterdam, Elsevier/Academic Press. EDWARDS, G. (2010). The day I was crucified: as told by Jesus the Christ. 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