Difference between revisions of "Part:BBa K299812:Experience"

No review score entered. yunting and xinyi from SPSingapore 2015

In general, the sequence of the construct we've received differs significantly from the documented nucleotide sequence. In addition, the full suffix is missing within the construct we received (only Pstl is present). This resulted in our inability to sequence the construct using prefix&suffix primers, or primers that were designed based on the documented nucleotide sequence. We had to use universal primers to sequence the first 1kb, then design new primers for the next kb, and so on, resulting in 3 staggered rounds of sequencing.

The sequencing results point us to a few major and potentially destructive problems:

Segment containing Invasin:

There is very little nucleotide similarity between the obtained sequence and the documented sequence on the website. Even the translated amino acid sequences have only 55% identity. According to the entry for BBa_K299810, the Invasin sequence contains the inv gene from Yersinia pestis obtained through horizontal gene transfer from Yersinia pseudotuberculosis. Attempts to determine the origin of the inv gene through BLAST shows a 100% match with the sequence from Yersinia enterocolitica" but partial match against that from Yersinia pseudotuberculosis.

In addition, the origin and function of the additional 300bp between the Invasin and Listerolysin fragment within the construct itself (just before the 2nd RBS) is not known to us. The size of the construct received is actually 4460bp (PstI site excluded) instead of 4146bp.

Furthermore, the documented nucleotide sequence for Invasin (part BBa_K177010) was found to be the reverse complement of the sequence of Invasin from the actual construct that we have received.

Segment containing Listerolysin:

The Listerolysin sequence documented online contains numerous mismatches when aligned with the sequence that we have obtained through sequencing, although there is 99% similarity in the translated amino acid sequence (perhaps the sequence had been codon-optimised, but documentation does not reflect this). The stop codon for Listerolysin is also missing.

Sequenced construct received:

File:BBa K299812-prime.txt