Composite

Part:BBa_K3407018:Design

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


Mini-3 endoribonuclease with araBAD promoter - RBS - and rrnBT1 terminator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 300
    Illegal XbaI site found at 673
    Illegal PstI site found at 527
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 300
    Illegal PstI site found at 527
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 300
    Illegal BglII site found at 309
    Illegal BamHI site found at 809
    Illegal XhoI site found at 818
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 300
    Illegal XbaI site found at 673
    Illegal PstI site found at 527
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 300
    Illegal XbaI site found at 673
    Illegal PstI site found at 527
    Illegal AgeI site found at 74
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 56

Design Notes

The gene was amplified by PCR from Bacillus subtilis strain 168, which has the ATCC6051 genome that is the same strain as NCCB 70064, also same as KTCC 1028. Mini-3 amino acid sequence was retrieved from UniProt (UniProt ID: O31418), introduced as a query in tBlastn from NCBI against B. subtilis ATCC 6051 genome and the resulting mini3 sequence (GenBank: CP011115.1) 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 Gibson Assembly. The primers used were the following:

M3-039 (mini3-Fw)

5’ - gaattcaaaagatcttttaagaaggagatatacatATGCTTGAATTTGATACGATAAAAGATTCTAAGCAG - 3’

M3-040 (mini3-Rv)

5’ - tttatttgatgcctggagatccttactcgagtttggatccTTAGTGGTGGTGGTGATGGTGGTCGCTTTTTACGCTGGTTTCCTCTGTTGCTGACTCATTTGTTTTCCTCC - 3’

Source

Genomic DNA from B. subtilis strain 168. Plasmid used pBbE8c backbone from BglBricks. pBbE8c-RFP was a gift from Jay Keasling (Addgene plasmid # 35269; http://n2t.net/addgene:35269; RRID:Addgene_35269) [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