Difference between revisions of "Part:BBa K2255006:Design"

Revision as of 16:28, 29 September 2017

EPS-Depolymerase (Rfc25)

Design Notes

Erwinia amylovora is gram-negative bacterium in the family Enterobacteria. This bacterium has a similar path of infection as Xylella Fastidiosa: Erwinia amylovora makes its entry into the host xylem and synthesize the extracellular polysaccharides (EPS) occluding the xylem vessels of the plant.

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, naturally present phages or the natural defence of the plant.

The first step was to run a BLAST using the EPS-Depolymerase from Erwinia amylovora (Q9G072_9CAUD) to see if an identified EPS-Depolymerase was present in phages against Xylella fastidiosa, but alas the results were negative. However, 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 contains two functions, one structural and one pectin lyase. Multiple articles showed that some phages that work on Xanthomonas can also work on Xylella fastidiosa and Vice versa. However, we can't be sure if the gene we chose can work because of the lack of documentation.

The protein is 852 amino acid which correspond to 2556 nucleobases, which is quite big, making it difficult to work with, so a process of trimming was done. Seeing as the domain we are interested in, the pectin lyase domain, spans 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 the relative conservation of this domain and if the excess were not aligned with the query sequence DIBBI_029. We found a majority of the alignments started with a gap of 78 amino acids in the N-terminus and 266 amino acids in the C-terminus so we chose to reduce the size of the protein to 510 amino acids, leading to 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 holds two restriction sites for EcoRI and xbaI and the suffix holds SpeI and PstI.

The sequence was retro translated to obtain the genomic sequence with the E.coli bias. ATGCAGATTCAGGAACCGGATGGCTTTAAATATATTGGCCGCGTGCCGAGCTTTGCGGCGCTGGTGAGCGTGGTGCCGGAAAAAGCGGGCGAACGCGTGATT GTGAGCGGCCATGTGGCGGGCAACGATTATGGCGGCGGCGTGTTTGTGGCGCGCGCGGGCAGCGTGGCGATTAACGATGGCGGCACCATTATGCCGGTGAAC AACAACTTTTATTGGCAGCGCCTGGTGGAAGATCCGGGCACCCTGGATGTGACCCATTTTGGCGCGAAACGCGATGGCGTGACCGATTGCGCGACCGCGTGC CTGGCGATGTGGAACTATACCCAGAGCCTGGGCGCGGGCGGCAGCATGATTGGCATTCAGTTTCCGGCGGGCGAATTTGCGGTGAGCAACATTGATATTAGC GCGAACTATGTGGGCAACTTTCGCCTGGTGGGCAAAGGCGTGGTGACCACCTTTGGCTATTTTCCGGCGACCCGCATTAAACTGATTGGCGCGGATAACCAG GCGGCGTTTAAAGTGCAGGCGCGCCGCAGCGAAATTGCGAACCTGCAGATTTATGGCCAGTATGAAGTGAAAGCGAACACCCGCGGCTTTTTTAAAAACACC TGCGTGAGCGGCCAGTATGTGCATGGCGTGAACTGGCGCAGCACCTATACCGGCGGCCCGATTTTTGATCTGATGGATACCCTGGATACCAAATTTAGCGAA TTTTATGCGAGCTATGTGTATGGCGGCGTGATTTATGGCGTGCCGAGCGGCAGCGAAAGCGGCAGCTGGGATCATCTGACCGCGATTGAACTGAGCAACTTT AACGTGCAGCGCTGCTATGGCAAACAGGCGTTTGATCTGCAGAAAAGCGGCCAGAGCTTTATTTATAACGGCTGGATTGAAAAAACCGATTTTCCGGGCGAT CTGAGCAACGGCCAGTGGATTATTCAGGGCCTGAGCATGGAAGATTGCGTGAACCCGCTGGATCTGACCTTTACCCGCGCGCAGCTGAGCCAGATTAACCTG CAGGGCACCAGCGCGCTGCGCTATGATAACCCGGATAAAAGCCGCCTGCTGAGCACCTATGAAATGGGCCGCAACCGCGTGGAAGCGTATGGCGCGCAGTTT TTTGGCAGCCTGAGCTATGATTATCTGAGCAGCCATTATCGCCTGAGCAACGCGACCGATAAAGCGGCGTGGTTTAACCTGGGCAAAATGATTGTGACCAAC CAGAACGATGCGAGCCGCATTCGCTTTTTTGGCGCGAACGGCCAGGCGAGCGTGCCGAGCGATCAGGGCGCGTTTGATAGCAACAACTTTGGCGGCGGCGAA TGCCTGCTGACCCTGCGCCGCGTGCCGGGCACCGGCACCCGCCAGGATTGCGCGATTGAAGTGCATGGCAACAGCCCGATTGCGGATATTCGCATTAGCCGC CCGTATGAAAACGATGTGGAAATTTATGTGCAGCTGAAACCGCAGTGCGGCTTTGTGAACGTGAGCCTGGAAACCAGCACCAACAGCCGCTTTGATAGCGGC

However, this sequence had three restriction sites for the endonuclease PSTI which is in conflict with the endonuclease we wanted to use 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 are found on the 555th, 855th and 1020th position with the conserved sequence of CTGCAG, CTG coding for a leucine and CAG coding for a glutamine. Thankfully, the genomic code is redundant so we can modify nucleobases without changing the translated protein. We chose to change the G of CTG into an A giving us CTA; this modification did not produce any restriction sites critical to our work eliminating the restriction site for PSTI.

The resulting sequence

ATGCAGATTCAGGAACCGGATGGCTTTAAATATATTGGCCGCGTGCCGAGCTTTGCGGCGCTGGTGAGCGTGGTGCCGGAAAAA GCGGGCGAACGCGTGATTGTGAGCGGCCATGTGGCGGGCAACGATTATGGCGGCGGCGTGTTTGTGGCGCGCGCGGGCAGCGTG GCGATTAACGATGGCGGCACCATTATGCCGGTGAACAACAACTTTTATTGGCAGCGCCTGGTGGAAGATCCGGGCACCCTGGAT GTGACCCATTTTGGCGCGAAACGCGATGGCGTGACCGATTGCGCGACCGCGTGCCTGGCGATGTGGAACTATACCCAGAGCCTG GGCGCGGGCGGCAGCATGATTGGCATTCAGTTTCCGGCGGGCGAATTTGCGGTGAGCAACATTGATATTAGCGCGAACTATGTG GGCAACTTTCGCCTGGTGGGCAAAGGCGTGGTGACCACCTTTGGCTATTTTCCGGCGACCCGCATTAAACTGATTGGCGCGGAT AACCAGGCGGCGTTTAAAGTGCAGGCGCGCCGCAGCGAAATTGCGAACCTACAGATTTATGGCCAGTATGAAGTGAAAGCGAAC ACCCGCGGCTTTTTTAAAAACACCTGCGTGAGCGGCCAGTATGTGCATGGCGTGAACTGGCGCAGCACCTATACCGGCGGCCCG ATTTTTGATCTGATGGATACCCTGGATACCAAATTTAGCGAATTTTATGCGAGCTATGTGTATGGCGGCGTGATTTATGGCGTG CCGAGCGGCAGCGAAAGCGGCAGCTGGGATCATCTGACCGCGATTGAACTGAGCAACTTTAACGTGCAGCGCTGCTATGGCAAA CAGGCGTTTGATCTACAGAAAAGCGGCCAGAGCTTTATTTATAACGGCTGGATTGAAAAAACCGATTTTCCGGGCGATCTGAGC AACGGCCAGTGGATTATTCAGGGCCTGAGCATGGAAGATTGCGTGAACCCGCTGGATCTGACCTTTACCCGCGCGCAGCTGAGC CAGATTAACCTACAGGGCACCAGCGCGCTGCGCTATGATAACCCGGATAAAAGCCGCCTGCTGAGCACCTATGAAATGGGCCGC AACCGCGTGGAAGCGTATGGCGCGCAGTTTTTTGGCAGCCTGAGCTATGATTATCTGAGCAGCCATTATCGCCTGAGCAACGCG ACCGATAAAGCGGCGTGGTTTAACCTGGGCAAAATGATTGTGACCAACCAGAACGATGCGAGCCGCATTCGCTTTTTTGGCGCG AACGGCCAGGCGAGCGTGCCGAGCGATCAGGGCGCGTTTGATAGCAACAACTTTGGCGGCGGCGAATGCCTGCTGACCCTGCGC CGCGTGCCGGGCACCGGCACCCGCCAGGATTGCGCGATTGAAGTGCATGGCAACAGCCCGATTGCGGATATTCGCATTAGCCGC CCGTATGAAAACGATGTGGAAATTTATGTGCAGCTGAAACCGCAGTGCGGCTTTGTGAACGTGAGCCTGGAAACCAGCACCAAC AGCCGCTTTGATAGCGGC

If all is well and done, we should be able to clone the gene and collect the protein with a molecular weight of 55.81 kD, which is 510 amino acids without taking into account the histidine tag.

Source

Genomic sequence of the phage vB_XveM_DIBBI

References