From 2009.igem.org

Revision as of 14:42, 21 October 2009

This is a copy from our blog. [http://igem.sdu.dk/?p=358 Original post].

1K we called it. High hopes we had for it. A fraud it was. A fraud that also goes under the name of [http://partsregistry.org/partsdb/get_part.cgi?part=BBa_J04450 psB1A3 with BBa_J04450 inserted]. Here we detail the unravelling of this fraud.

Colony-PCR

Throughout our work, our colony-pcr's turned up short, sometimes even shorter than it should ever be able to be.

If you run a PCR with VF2() and VR() the shortest amplicon should be 316bp but we kept seeing bands at around 250bp. We turned our minds inside out trying to figure out ways that our parts could insert wrongly to produce the lengths seen, multiple primer sites was one of our main differential hypotheses.

Sequencing

Near-simultaneously we received sequencing data from the samples we had the most faith in and discovered this comment on the [http://partsregistry.org/Part:pSB1A3:Experience partsregistry]:

http://igem.sdu.dk/wp-content/uploads/comment.jpg

Following up on this information i tried to confirm that our plasmid was indeed psB1A2, but overworked and stressed out i initially couldn't make the evidence conclusive and our samples had already been sent to sequencing so i thought i'd try again later.

"><img class="size-full wp-image-362" title="psb1a3-distances" src="http://igem.sdu.dk/wp-content/uploads/psb1a3-distances.png" alt="Screenshot of <a href=" width=" mce_href=" height="164" /></a>Really it should be 196, i forgot to add the pre- and suffix lenghts *blush*. However, the point still stands, and it would be fantastic if we (read: someone) could implement a way for the registry to use the sequencing data, not just for the target part but also for the plasmid it is supposed to be in.

Then our own sequencing arrived and we tried aligning the sequence of our sample (minus insert) against pSB1A3 and pSB1A2. It clearly showed that the backbone we extracted from well 1K, supposedly psB1A3 with J04450, did not contain the 78 bp immediately following the suffix that differentiates A3 from A2.

Here's the alignments:

[caption id="attachment_370" align="alignleft" width="300" caption="BB sequence aligned against pSB1A2 expected sequence"]<a href="http://igem.sdu.dk/wp-content/uploads/bb-sekventeret-sammenligning-med-psb1a2.jpg"><img class="size-medium wp-image-370 " title="bb-sekventeret-sammenligning-med-psb1a2" src="http://igem.sdu.dk/wp-content/uploads/bb-sekventeret-sammenligning-med-psb1a2-300x123.jpg" alt="BB sequence against pSB1A2 expected sequence" width="300" height="123" /></a>[/caption]

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