Device

Part:BBa_K1137009

Designed by: Matthew Deyell   Group: iGEM13_Paris_Bettencourt   (2013-09-23)

sRNA anti Kan

This complex part contains everything necessary to express small RNA to inhibit expression of the Kanamycin resistance cassette encoding Aminoglycoside N6'-acetyltransferase. The biobrick contains a Pr promoter, target sRNA, a scaffold derived from MicC lacking the ompC binding sequence, and a T1/TE terminator. The scaffold forms the RNA secondary structure while the target sRNA of the sequence CGTTTCCCGTTGAATATGGCT binds to the target sequence of ATGAGCCATATTCAACGGGAAACG including the start codon and the first 24 bp of the kanamycin ORF within the mRNA. It is vital that the sequences are complementary for proper repression to occur. If there are point mutations within a copy of the kanamycin resistance cassette at this location, or a different kanamycin cassette is targeted the biobrick will not function correctly.

Anti-Kan_PR.png

We used a phage vector bearing the sRNA anti-Kan we designed to infect a population of kanamycin resistant cells (kanamycin resistance comes from commercial plasmid pCOLA Duet). By applying a sequential killing strategy described here : http://2013.igem.org/Team:Paris_Bettencourt/Project/Sabotage, we efficiently killed 99,87% of the bacterial population (kanamycin concentration 1000ug/ml).

PBsurvivingkan3.png

We witness a shift in the minimum inhibitory concentration of Kanamycin from 400ug/ml in Kanamycin Resistance E. coli to <12.5 ug/ml, the same MIC that we observed in WT E. coli.

Kan_sRNA_MIC.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 153
    Illegal XhoI site found at 159
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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