Difference between revisions of "Part:BBa K371015:Design"

 
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<partinfo>BBa_K371015 short</partinfo>
 
<partinfo>BBa_K371015 short</partinfo>
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===Design Notes===
 
===Design Notes===
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In order to isolate pduA from the Citrobactor freundii genome, following primers has been designed to make it a standard Biobrick.
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The present BioBrick assembly standard RFC does not support Protein fusion. In our project, we propose a new BioBrick assembly standard BBFRFC53---MetaPart Assembly Standard, which extending RFC 10 to Enable Scarless Protein Fusion with Type IIS Restriction Enzyme EarI and SapI.
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In the previous literature，most of research work on pdu BMC(or pdu MCP) focus on ''Citrobacter Freundii'' and ''Salmonella enterica''. Taking all of the factors together, we choose Citrobacter Freundii as the orgin of gene in our project. Because the whole genome of Citrobacter Freundii has not been decoded yet, we use the reference sequence sent to NCBI by Martin J.Warren group(GenBank: AM498294.1).
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In order to make the new parts compatible to new standard, we firstly check the sequence of pduU, which we get from GenBank(GenBank: AM498294.1), with the new four restriction enzymes site EarI, SapI,SacI and BglII. Luckily, none of these site were find in pduU. Then we use the following primers to get the pduU from BBa_K371001, wichi consist of natural pduTUV transcription unit. The final part consist of cds of pduU with meta-prefix and meta-suffix in its sides
  
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<html>
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<div style="padding-left:25px;background-color:#FFFACD">
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<p>forward primer:5'-GTTTCTCTTCAATGGAAAGACAACCCACCACGGATCGTATG-3'</p>
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<p>rreverse primer:5'-GTTTCTCTTCAACCTGTCCGGGTGATGGGACAGGCGGTAAAA-3'</p>
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</div>
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</html>
  
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The sequence results of pduU are different from the sequence we found on Genebank. Some silent mutation locate in the sequence with no special spacial rule. We suspect that the strain we used is not same with the papar, maybe even belong to different strain of ''Citrobacter Freundii''. Moreover the proterin sequence alignment of pduU in ''Citrobacter Freundii'',''Salmonella enterica'' and ours shows that the 'mutation' locate in the nonconserved site. Thus we decide to continue our experement with the gene pduU.
  
 
===Source===
 
===Source===

Latest revision as of 07:24, 27 October 2010

pduU


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 227
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The present BioBrick assembly standard RFC does not support Protein fusion. In our project, we propose a new BioBrick assembly standard BBFRFC53---MetaPart Assembly Standard, which extending RFC 10 to Enable Scarless Protein Fusion with Type IIS Restriction Enzyme EarI and SapI.

In the previous literature，most of research work on pdu BMC(or pdu MCP) focus on Citrobacter Freundii and Salmonella enterica. Taking all of the factors together, we choose Citrobacter Freundii as the orgin of gene in our project. Because the whole genome of Citrobacter Freundii has not been decoded yet, we use the reference sequence sent to NCBI by Martin J.Warren group(GenBank: AM498294.1).

In order to make the new parts compatible to new standard, we firstly check the sequence of pduU, which we get from GenBank(GenBank: AM498294.1), with the new four restriction enzymes site EarI, SapI,SacI and BglII. Luckily, none of these site were find in pduU. Then we use the following primers to get the pduU from BBa_K371001, wichi consist of natural pduTUV transcription unit. The final part consist of cds of pduU with meta-prefix and meta-suffix in its sides

forward primer:5'-GTTTCTCTTCAATGGAAAGACAACCCACCACGGATCGTATG-3'

rreverse primer:5'-GTTTCTCTTCAACCTGTCCGGGTGATGGGACAGGCGGTAAAA-3'

The sequence results of pduU are different from the sequence we found on Genebank. Some silent mutation locate in the sequence with no special spacial rule. We suspect that the strain we used is not same with the papar, maybe even belong to different strain of Citrobacter Freundii. Moreover the proterin sequence alignment of pduU in Citrobacter Freundii,Salmonella enterica and ours shows that the 'mutation' locate in the nonconserved site. Thus we decide to continue our experement with the gene pduU.

Source

Citrobactor freudii, the strain was bought from NITE Biological Resource Center (NBRC). NBRC NO. is 12681. History:IFO 12681 <- Ajinomoto Co., Inc. (AJ 2619) <- ATCC 8090 <- C.H. Werkm

References