Revision as of 14:11, 13 June 2017

EPS Depolymerase

To combat the problem that is Xylella fastidiosa we firstly started searching for natural solutions against this bacterium and surely, we found bacteriophages that loved destroying this bacterium.

In our researcher, a modified phage would not be possible because of restriction in spreading modified organism able to replicate in the wild. Therefore, a non-lytic and non-lysogenic phage was borne using fibrous phage M-13 with a modification of its genome in the objective of being modular and flexible to use against varies pathogens. The phages against Xylella fastidiosa have a devious way to infect the bacterium by cleaving the sugar bond in the biofilm then link on the receptors pili type IV on the membrane that will sets off a series of action leading to the injection of the bacteriophage genome in the bacterium triggering the life cycle of the phage. A number of articles described the possibility of using these phages to eradicate Xylella Fastidiosa but in my mind, that was an endeavor bigger than my capacities. One article talked about the EPS-Depolymerase as a potential tool for control of fire blight a disease caused by Erwinia amylovora.

Erwinia amylovora is gram-negative bacterium in the family Enterobacteria. This bacterium have a similar path of infection as Xylella Fastidiosa, Erwinia amylovora makes its entry into its host xylem synthesis the extracellular polysaccharides (EPS). EPS helps bacterial avoid plant defenses, and clog the host vascular system.

This information narrowed my research in the direction of determining enzymes capable of degrading the biofilm of Xylella Fastidiosa rendering it more susceptible to antibiotic or naturally present phages.

First step was to run a blast using the EPS-Depolymerase from Erwinia amylovora (Q9G072_9CAUD) to see if an EPS-Depolymerase was present in phages against Xylella fastidiosa, but alas the results where negative but a potential candidate arose.

Putative tail fiber protein/putative EPS Depolymerase (DIBBI_029) is an enzyme of 852 amino acids, from the organism Xanthomonas phage vB_XveM_DIBBI which contain two function one structural and one pectin lyase. Xanthomonas is a close relative of Xylella fastidiosa, multiple articles shows that some phages that work on Xanthomonas can work on Xylella fastidiosa and Vis versa. However, we can be sure if the gene chosen can work or not because of the lack of documentation.

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The protein is 852 amino acid with is translated to 2556 nucleobases is quite big making it difficult to work with so a process of trimming was donne. Seeing as the domain we are interested in pectin lyase start from the 139th to 426th amino acid it is possible to remove the excess amino acids without harming the functionality and specificity of the enzyme. A Blast against NR was done to see if this domain is highly conserved and if the excess were not aligned with the the query sequence DIBBI_029. We found a majority of the alignment started with a gap of 78 amino acids in N-terminus and 266 amino acids in C-terminus so we obted to reduce the size of the protein to 510 amino acids a 342 amino acid reduction.

image

The protein is 852 amino acid with is translated to 2556 nucleobases is quite big making it difficult to work with so a process of trimming was donne. Seeing as the domain we are interested in pectin lyase start from the 139th to 426th amino acid it is possible to remove the excess amino acids without harming the functionality and specificity of the enzyme. A Blast against NR was done to see if this domain is highly conserved and if the excess were not aligned with the the query sequence DIBBI_029. We found a majority of the alignment started with a gap of 78 amino acids in N-terminus and 266 amino acids in C-terminus so we obted to reduce the size of the protein to 510 amino acids a 342 amino acid reduction.

Giving us the sequence present:

MQIQEPDGFKYIGRVPSFAALVSVVPEKAGERVIVSGHVAGNDYGGGVFVARAGSVAINDGGTIMPVNNNFYWQRLVEDPGTLDVTHFGAKRDGVTDCATAC LAMWNYTQSLGAGGSMIGIQFPAGEFAVSNIDISANYVGNFRLVGKGVVTTFGYFPATRIKLIGADNQAAFKVQARRSEIANLQIYGQYEVKANTRGFFKNT CVSGQYVHGVNWRSTYTGGPIFDLMDTLDTKFSEFYASYVYGGVIYGVPSGSESGSWDHLTAIELSNFNVQRCYGKQAFDLQKSGQSFIYNGWIEKTDFPGD LSNGQWIIQGLSMEDCVNPLDLTFTRAQLSQINLQGTSALRYDNPDKSRLLSTYEMGRNRVEAYGAQFFGSLSYDYLSSHYRLSNATDKAAWFNLGKMIVTN QNDASRIRFFGANGQASVPSDQGAFDSNNFGGGECLLTLRRVPGTGTRQDCAIEVHGNSPIADIRISRPYENDVEIYVQLKPQCGFVNVSLETSTNSRFDSG

Our primary cloning medium is the plasmid pSB1C3, we chose RFC 25 as the prefix and suffix for the sequence and the E.coli promoter

To purify the protein we added a histidine tag at 3’, which enable a chromatography on a nickel column. The prefix and suffix are special sequence designed with restriction sites to flank both ends of the sequence we are trying to modify. the prefix hold two restriction sites for EcoRI and xbaI, the suffix holds SpeI and PstI.

The sequence was retro translated to obtain the genomic sequence with the E.coli bias. ATGCAGATTCAGGAACCGGATGGCTTTAAATATATTGGCCGCGTGCCGAGCTTTGCGGCGCTGGTGAGCGTGGTGCCGGAAAAAGCGGGCGAACGCGTGATTGTGAGCGGCCATGTGGCGGGCAACGATTATGGCGGCGGCGTGTTTGTGGCGCGCGCGGGCAGCGTGGCGATTAACGATGGCGGCACCATTATGCCGGTGAACAACAACTTTTATTGGCAGCGCCTGGTGGAAGATCCGGGCACCCTGGATGTGACCCATTTTGGCGCGAAACGCGATGGCGTGACCGATTGCGCGACCGCGTGCCTGGCGATGTGGAACTATACCCAGAGCCTGGGCGCGGGCGGCAGCATGATTGGCATTCAGTTTCCGGCGGGCGAATTTGCGGTGAGCAACATTGATATTAGCGCGAACTATGTGGGCAACTTTCGCCTGGTGGGCAAAGGCGTGGTGACCACCTTTGGCTATTTTCCGGCGACCCGCATTAAACTGATTGGCGCGGATAACCAGGCGGCGTTTAAAGTGCAGGCGCGCCGCAGCGAAATTGCGAACCTGCAGATTTATGGCCAGTATGAAGTGAAAGCGAACACCCGCGGCTTTTTTAAAAACACCTGCGTGAGCGGCCAGTATGTGCATGGCGTGAACTGGCGCAGCACCTATACCGGCGGCCCGATTTTTGATCTGATGGATACCCTGGATACCAAATTTAGCGAATTTTATGCGAGCTATGTGTATGGCGGCGTGATTTATGGCGTGCCGAGCGGCAGCGAAAGCGGCAGCTGGGATCATCTGACCGCGATTGAACTGAGCAACTTTAACGTGCAGCGCTGCTATGGCAAACAGGCGTTTGATCTGCAGAAAAGCGGCCAGAGCTTTATTTATAACGGCTGGATTGAAAAAACCGATTTTCCGGGCGATCTGAGCAACGGCCAGTGGATTATTCAGGGCCTGAGCATGGAAGATTGCGTGAACCCGCTGGATCTGACCTTTACCCGCGCGCAGCTGAGCCAGATTAACCTGCAGGGCACCAGCGCGCTGCGCTATGATAACCCGGATAAAAGCCGCCTGCTGAGCACCTATGAAATGGGCCGCAACCGCGTGGAAGCGTATGGCGCGCAGTTTTTTGGCAGCCTGAGCTATGATTATCTGAGCAGCCATTATCGCCTGAGCAACGCGACCGATAAAGCGGCGTGGTTTAACCTGGGCAAAATGATTGTGACCAACCAGAACGATGCGAGCCGCATTCGCTTTTTTGGCGCGAACGGCCAGGCGAGCGTGCCGAGCGATCAGGGCGCGTTTGATAGCAACAACTTTGGCGGCGGCGAATGCCTGCTGACCCTGCGCCGCGTGCCGGGCACCGGCACCCGCCAGGATTGCGCGATTGAAGTGCATGGCAACAGCCCGATTGCGGATATTCGCATTAGCCGCCCGTATGAAAACGATGTGGAAATTTATGTGCAGCTGAAACCGCAGTGCGGCTTTGTGAACGTGAGCCTGGAAACCAGCACCAACAGCCGCTTTGATAGCGGC

However, this sequence had three restriction sites for the endonuclease PSTI which is in conflict with the endonuclease we will be using to modify and insert the gene in the plasmid. Keeping these sites will cleave the gene in three separate places losing all functionality. The three sites found on the 555th, 855th and 1020th position with the conserved sequence of CTGCAG, CTG code for a leucine and CAG code for a glutamine. Thankfully, the genomic code is redundant so we can modify nucleobases without changing the protein translated. We chose to change the G of CTG to an A giving us CTA this modification did not produce any restriction sites critical to our work eliminating the restriction site for PSTI.

Resulting in the sequence as present

ATGCAGATTCAGGAACCGGATGGCTTTAAATATATTGGCCGCGTGCCGAGCTTTGCGGCGCTGGTGAGCGTGGTGCCGGAAAAAGCGGGCGAACGCGTGATTGTGAGCGGCCATGTGGCGGGCAACGATTATGGCGGCGGCGTGTTTGTGGCGCGCGCGGGCAGCGTGGCGATTAACGATGGCGGCACCATTATGCCGGTGAACAACAACTTTTATTGGCAGCGCCTGGTGGAAGATCCGGGCACCCTGGATGTGACCCATTTTGGCGCGAAACGCGATGGCGTGACCGATTGCGCGACCGCGTGCCTGGCGATGTGGAACTATACCCAGAGCCTGGGCGCGGGCGGCAGCATGATTGGCATTCAGTTTCCGGCGGGCGAATTTGCGGTGAGCAACATTGATATTAGCGCGAACTATGTGGGCAACTTTCGCCTGGTGGGCAAAGGCGTGGTGACCACCTTTGGCTATTTTCCGGCGACCCGCATTAAACTGATTGGCGCGGATAACCAGGCGGCGTTTAAAGTGCAGGCGCGCCGCAGCGAAATTGCGAACCTACAGATTTATGGCCAGTATGAAGTGAAAGCGAACACCCGCGGCTTTTTTAAAAACACCTGCGTGAGCGGCCAGTATGTGCATGGCGTGAACTGGCGCAGCACCTATACCGGCGGCCCGATTTTTGATCTGATGGATACCCTGGATACCAAATTTAGCGAATTTTATGCGAGCTATGTGTATGGCGGCGTGATTTATGGCGTGCCGAGCGGCAGCGAAAGCGGCAGCTGGGATCATCTGACCGCGATTGAACTGAGCAACTTTAACGTGCAGCGCTGCTATGGCAAACAGGCGTTTGATCTACAGAAAAGCGGCCAGAGCTTTATTTATAACGGCTGGATTGAAAAAACCGATTTTCCGGGCGATCTGAGCAACGGCCAGTGGATTATTCAGGGCCTGAGCATGGAAGATTGCGTGAACCCGCTGGATCTGACCTTTACCCGCGCGCAGCTGAGCCAGATTAACCTACAGGGCACCAGCGCGCTGCGCTATGATAACCCGGATAAAAGCCGCCTGCTGAGCACCTATGAAATGGGCCGCAACCGCGTGGAAGCGTATGGCGCGCAGTTTTTTGGCAGCCTGAGCTATGATTATCTGAGCAGCCATTATCGCCTGAGCAACGCGACCGATAAAGCGGCGTGGTTTAACCTGGGCAAAATGATTGTGACCAACCAGAACGATGCGAGCCGCATTCGCTTTTTTGGCGCGAACGGCCAGGCGAGCGTGCCGAGCGATCAGGGCGCGTTTGATAGCAACAACTTTGGCGGCGGCGAATGCCTGCTGACCCTGCGCCGCGTGCCGGGCACCGGCACCCGCCAGGATTGCGCGATTGAAGTGCATGGCAACAGCCCGATTGCGGATATTCGCATTAGCCGCCCGTATGAAAACGATGTGGAAATTTATGTGCAGCTGAAACCGCAGTGCGGCTTTGTGAACGTGAGCCTGGAAACCAGCACCAACAGCCGCTTTGATAGCGGC

If all well and done, we should be able to clone the gene and recuperate the protein with a Molecular weight of 55.81 KD and 510 amino acids without taking into account the histidine tag.