Difference between revisions of "Part:BBa K299812:Experience"
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<I>'''yunting''' from SPSingapore 2015</I>
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<I>'''yunting and xinyi''' from SPSingapore 2015</I>
 
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The sequence of the part was significantly different as was documented. Our team had trouble sequencing it initially as the full suffix sequence was absent, and primers that were designed based on the documented nucleotide sequence didn't work. We had to use universal primers to sequence the first 1kb, then design new primers for the next kb. Thus we had 3 staggered rounds of sequencing.  
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In general, the  sequence of the construct we've received differs significantly from the documented nucleotide sequence.  
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Additionally, the full suffix is missing within the construct we received (only Pstl is present). As such, 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.  
  
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The sequencing results point us to a few major and potentially destructive problems:
  
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'''''Segment containing Invasin:'''''
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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 with each other.
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The size of the whole construct received is actually 4460bp (PstI site excluded) instead of 4146bp. The origin and function of the additional 300bp between the Invasin and Listerolysin fragment (just before the 2nd RBS) is not known to us. Moreover, BBa_K299810 (the basic part BBa_K299812 is assembled from)  is stated to contain the ''inv'' gene from ''Yersinia pestis'', which in turn was obtained via horizontal gene transfer from ''Yersinia pseudotuberculosis''. Attempts to determine the origin of the ''inv'' gene through BLAST shows a 100% match with a nucleotide sequence from ''Yersinia enterocolitica'' (GenBank: CP009846.1) but the best match from ''Yersinia pseudotuberculosis'' sequences (GenBank: CCH50685.1) only showed partial matches.
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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 we have received.
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'''''Segment containing Listerolysin:'''''
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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.
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'''''Sequencing result:'''''
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[[File:BBa_K299812-prime.txt]]
  
 
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Latest revision as of 15:52, 18 September 2015

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K299812

User Reviews

UNIQa7a0494f6fff4fe2-partinfo-00000000-QINU

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. Additionally, the full suffix is missing within the construct we received (only Pstl is present). As such, 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 with each other.

The size of the whole construct received is actually 4460bp (PstI site excluded) instead of 4146bp. The origin and function of the additional 300bp between the Invasin and Listerolysin fragment (just before the 2nd RBS) is not known to us. Moreover, BBa_K299810 (the basic part BBa_K299812 is assembled from) is stated to contain the inv gene from Yersinia pestis, which in turn was obtained via horizontal gene transfer from Yersinia pseudotuberculosis. Attempts to determine the origin of the inv gene through BLAST shows a 100% match with a nucleotide sequence from Yersinia enterocolitica (GenBank: CP009846.1) but the best match from Yersinia pseudotuberculosis sequences (GenBank: CCH50685.1) only showed partial matches.

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 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.

Sequencing result:

File:BBa K299812-prime.txt

UNIQa7a0494f6fff4fe2-partinfo-00000002-QINU

UNIQa7a0494f6fff4fe2-partinfo-00000003-QINU