Team:UNIPV-Pavia/Debug

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Our new parts

Re-built existing parts (BBa_our part code/BBa_existing part code)

Existing parts from the Registry

Existing parts sequence debugging:

• BBa_T9002 - BBa_K131009 - BBa_K131010 - BBa_K116001 - BBa_K116002 - BBa_P0412 - BBa_K112405 - BBa_K101017 - BBa_I746902 - BBa_I746903 - BBa_K112808 - BBa_F2620

During this summer, we sequenced and carefully analyzed sequencing results of some existing BioBrick parts. Some of them were not present in DNA Distribution, so they have been sent us by iGEM Headquarters.

BBa_T9002

The UNIPV-Pavia iGEM team sequenced this part and found that it was completely confirmed, while iGEM QC 2009 DNA distribution classified it as "inconsistent". DNA was resuspended from well 9A, kit plate 2, transformed in TOP10 E. coli and amplified inoculating a single colony from the grown LB agar plate in LB medium. Finally DNA has been miniprepped from the grown culture and sent to a BMR Genomics (Padova, Italy) for sequencing.

BBa_K131009

After the alignment of this BioBrick sequence from Quality Control with the theoretical sequence, some difformities have been reported (sequencing results have been taken from iGEM QC): i) there is a non-silent point mutation in nucleotide 2142 which changes an amino acid of celB gene; ii) non-standard prefix (even if all the restriction sites are correct); iii) non-standard suffix (even if all the restriction sites are correct).

BBa_K131010

This part has been classified as "LONG PART" by iGEM QC, but we noticed that the 3' end of this part is not correct: it does not contain BBa_K131009.

BBa_K116001

The UNIPV-Pavia iGEM 2009 team requested this part from iGEM Headquarters, but sequence was not correct: actually, the part contained in the plasmid was BBa_K116002 and it also has an additional undocumented "c" nucleotide at the end of nhaA promoter (found after sequencing). Because BBa_K116002 is the measurement system of K116001 BioBrick, we used it to characterize the activity of nhaA promoter.

BBa_K116002

The UNIPV-Pavia iGEM 2009 team requested this part from iGEM Headquarters, but sequence was not correct: actually, the part contained in the plasmid was BBa_J33204 (found after sequencing).

BBa_P0412

This part was not present in 2009 DNA Distribution and no quality control had been performed. The UNIPV-Pavia requested this part to iGEM Headquarters, sequenced it and found that sequence was correct.

BBa_K112405

This part was not present in 2009 DNA Distribution and no quality control had been performed. The UNIPV-Pavia requested this part to iGEM Headquarters, sequenced it and found that sequence was correct.

BBa_K101017

This part was not present in 2009 DNA Distribution and no quality control had been performed. The UNIPV-Pavia requested this part to iGEM Headquarters, sequenced it, but found that the sequence was inconsistent: the sequencing partner (BMR Genomics) found a very bad chromatogram.

BBa_I746902

This part was not present in 2009 DNA Distribution and no quality control had been performed. The UNIPV-Pavia requested this part to iGEM Headquarters, sequenced it and found that the covered part of the sequence was correct (almost all the sequence was covered).

BBa_I746903

This part was not present in 2009 DNA Distribution and no quality control had been performed. The UNIPV-Pavia requested this part to iGEM Headquarters, sequenced it and found that the RBS was BBa_B0030 instead of BBa_B0034. All the rest of the sequence was confirmed.

BBa_K112808

After the alignment of this BioBrick sequence from 2009 DNA Distribution with the theoretical sequence, many difformities have been reported (sequencing results have been taken from iGEM QC): i) the alignment showed several gaps in the scars; ii) there is a silent point mutation in holin gene; iii) the transcriptional terminator at the end of the sequence is actually K112710, not B0010. However, the part should work properly because the regulatory parts and the amino acid sequence of the genes are correct. This has also been confirmed by functional tests.

BBa_F2620

This part was inconsistent in 2009 DNA Distribution. The UNIPV-Pavia requested this part to iGEM Headquarters, sequenced it and found that sequence was correct.

All these comments are also reported in the Experience pages of the relative BioBrick parts in the Registry web site.