Composite

Part:BBa_K3407019:Design

Designed by: Javier Navarro Delgado   Group: iGEM20_TUDelft   (2020-10-23)


YmdB regulator of RNAseIII in E. coli with tetA/tetR promoters - RBD - T1 terminator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 59
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 59
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 59
    Illegal BglII site found at 68
    Illegal BamHI site found at 673
    Illegal XhoI site found at 682
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 59
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 59
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The gene was amplified by PCR from E.coli Bl21(DE3) genome as a template. YmdB amino acid sequence was taken from Uniprot (Uniprot ID: P0A8D6), it was introduced as a query in tBlastn from NCBI, against Escherichia coli BL21(DE3) genome and the resulting YmdB gene sequence (GenBank: AM946981.2) was used as a template for the design of the primers. Primers were designed to have a melting temperature of 59-60 ºC. Overhangs with BglBricks Prefix and Suffix were added, along with a His-Tag in the Reverse primer for a C-terminal-tagged protein. Designed to be cloned through Assembly. The primers used were the following:

M3-037 (YmdB - Fw):

5’ - gaattcaaaagatcttttaagaaggagatatacatATGAAAACGCGTATTCATGTTGTGC - 3’

M3-038 (YmdB - Rv):

5’- tttatttgatgcctggagatccttactcgagtttggatccttaGTGATGGTGGTGATGATGGTGGTGATGGTGACCTGTACTTCCTGTTTCATCTCCTTGTTGGGTAAGGAGTC - 3’

Source

Plasmid used pBbA2k backbone from BglBricks. pBbA2k-RFP was a gift from Jay Keasling (Addgene plasmid # 35327 ; http://n2t.net/addgene:35327 ; RRID:Addgene_35327) [1]. Please refer to the Addgene page for more information about licences associated with the use of the plasmid.

References

Ordered List

  1. BglBrick vectors and datasheets: A synthetic biology platform for gene expression. Lee TS, Krupa RA, Zhang F, Hajimorad M, Holtz WJ, Prasad N, Lee SK, Keasling JD. J Biol Eng. 2011 Sep 20;5:12. 10.1186/1754-1611-5-12 PubMed 21933410