Part:BBa_K299812:Experience

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UNIQe5751f05a37345c4-partinfo-00000000-QINU

No review score entered. yunting and xinyi from SPSingapore 2015

In general, the full suffix is missing within the construct that we have received from the headquarters (only Pstl is present). This result in the inability to sequence the construct using primers that were designed based on the documented nucleotide sequence. Instead, We had to use universal primers to sequence the first 1kb, then design new primers for the next kb, resulting in 3 staggered rounds of sequencing. The sequencing results point us to a few major and potentially destructive problem: Segment containing Invasin: There is very little nucleotide similarity between the obtained sequence and the documented sequence on the website. The provided nucleotide sequence given for Invasin was found to be the reverse complement of the sequence of Invasin from the actual construct that we have received. Furthermore, 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. The origin and function of the additional 300bp between the Invasin and Listerolysin fragment within the construct itself (on top of the already existing RBS) is not known to us. Segment containing Listerolysin: The Listerolysin sequence provided online conatins numerous mismatch when aligned with the sequence that we have obtained through sequencing even though there is 99% similarity in the translated amino acid sequence.

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