Coding

Part:BBa_K371008:Design

Designed by: Yang Zhang   Group: iGEM10_USTC   (2010-10-08)

pduA


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 288
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 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 [http://bbf.openwetware.org/RFC.html#BBF_RFC_53:_USTC_MetaPart_Assembly_Standard_--_Extending_RFC_10_to_Enable_Scarless_Protein_Fusion_with_Type_IIS_Restriction_Enzyme_EarI_and_SapI|BBF RFC 53]---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 pduA, which we get from GenBank(GenBank: AM498294.1), with the new four restriction enzymes site EarI, SapI,SacI and BglII. Luckily, none of these sites were find in pduA. Then we use the following primers to get the pduA from BBa_K371001, which consist of natural pduAB transcription unit. The final part consist of coding of pduA with meta-prefix and meta-suffix in its sides. Here is a workflow depicting the process of making this parts. The new plasmid backbone BBa_K371051has a single base-pair mutation compared with pSB1C3 to eliminate the potential SacI site. The parts BBa_K371053 is a short DNA sequence to make the plasmid comply with RFC 53.


forward primer:5'-GTTTCTCTTCAATGCAACAAGAAGCGTTAGG-3'

reverse primer:5'-GTTTCTCTTCAACCGCTAATTCCCTTCGGTAAGATTT-3'

PduA Biobrick construction workflow based on RFC 53 standard.png


The sequence results of pduA 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 pduA 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 pduA.

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. Werkman

References