Composite

Part:BBa_K3505044

Designed by: Asteria Tsapadikou   Group: iGEM20_Thessaly   (2020-10-26)


pAndersonJ23115:TetO:RBS-eCFP -terminator

eCFP BBa_K3505020uder control of a constitutive promoter (andersonJ23115 with a tet operator) BBa_K3505014.

Fig.1:The Level A construct AndersonJ23115:TetO:rbs-eCFP-Terminator

Usage and Biology

This Trancriscription Unit (TU) is continuesly activated exressing the eCFP protein as a reporter. The Tet operator that is downsteam the anderson exists for the TetR regulated inhibition.

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BtgZI, BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in a1R BBa_K3505008 and has overhangs compatible for GoldenBraid cloning.

Verification of cloning

Fig.2:(U=Uncut C=Cut) (U=Uncut , C= Cut) Restriction digestion of TE: AndersonJ23115:Tet0-ECFP-double terminator(C1-C 4) with : EcoRV(C1-C2) , Expected bands : 2847+ 954 bp ,Positive result: C1,C2

Experimental Use and Experience

This part ,AndersonJ23114:TetO:RBS-ECFP-Double terminator , is very important for our engineering success , as it is our reporter module that is regulated from LacI. In the presence of LacI the transcription unit is blocked and there is no-fluoresence signal. This is the visualisation behind the NOT-Gate device. When we completed the cloning experiments, we immediately started plate-reader assays in order to validate the expression of ECFP , in absence of LacI. This chart shows the expression of ECFP, after 16h of incubation at 37oC , using M9 Medium.

Measurements are the average of 9 total replicates (3 biological replicates and 3 technical replicates per biological replicate). Error bars represent standard deviation of biological replicates

Fig.3:Validation that J23115-TetO BBa_K3505044 and J23115-LacO fusion promoters are able to drive expression of ECFP.Expression of ECFP, after 16h of incubation at 37oC , using M9 Medium.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 11
    Illegal NheI site found at 34
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

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