Reporter

Part:BBa_M36016:Design

Designed by: Kara Helmke Rogers   Group: Stanford BIOE44 - S11   (2017-04-19)


Thiosulfate Reporter (ThsR)


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 999
    Illegal SpeI site found at 863
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 999
    Illegal SpeI site found at 863
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 999
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 999
    Illegal SpeI site found at 863
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 999
    Illegal SpeI site found at 863
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

In designing our plasmids, we wanted to minimize replication competition, so we used one high copy ORI and one low copy ORI. The plasmids also have two different antibiotic resistances, allowing us to select for bacteria with both plasmids. pThsRR, utilizes a pJ-Amp-low backbone, which was acquired from DNA2.0. This backbone instead contains a low copy origin of replication and Ampicillin resistance genes to ensure transformation of both plasmids and minimal replication competition between plasmids. ThsR expression is driven by DNA2.0’s pRha rhamnose-inducible promoter (Parts registry number: BBa_K914003) and the same strong RBS as ThsS. The codon-optimized sequence for ThsR was identified and copied to our plasmid from pKD184, which was also deposited into Addgene by the same authors.11 A C-terminal FLAG tag was added to ThsR (Parts registry number: BBa_M10019), and transcription is terminated by the same Term_PhageT7 terminator, both coming from DNA2.0. On this same plasmid, we added the VixenPurple chromoprotein, obtained from DNA2.0, driven by the PphsA342 promoter, which is activated by ThsR binding. This reporter assembly was added in an anti-parallel fashion to the ThsR expression cassette, in order to minimize any errant transcription of DNA. The sequence for PphsA342 was located in the supplementary materials of the Tabor paper and inserted upstream of the DNA2.0 Strong RBS and VixenPurple protein.5 Termination is mediated by the same Term_PhageT7 terminator.

Source

Term_PhageT7: BBa_M50060
FLAG: BBa_T2004
ThsR: BBa_M50100
Strong RBS: BBa_M50080
pRha rhamnose inducible: BBa_K914003
Promoter_PphsA342: BBa_M50109
Strong RBS: BBa_M50080
VixenPurple: BBa_M50110
Term_Phage T7: BBa_M50060

References