Revision as of 00:37, 28 October 2020

pAndersonJ23115:lacO:RBS-eCFP -terminator

eCFP BBa_K3505020uder control of a constitutive promoter (andersonJ23115 with a lac operator) BBa_K2924013.

Fig.1:J23115-eCFP-Terminator

Usage and Biology

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

Design Notes

The coding sequence was domesticated . We removed BsmBI ,BsaI , BpiI sites in order to be compatible with GoldenBraid and MoClo. The sequence is cloned in both a1R BBa_K3505008and a2 BBa_K3505009 and has overhangs compatible for GoldenBraid cloning.

Verification of cloning

Cloned in alpha 2

Fig.2:(U=Uncut C=Cut) (U=Uncut , C= Cut) Restriction digestion of LE: AndersonJ23115:Lac0-EGFP-double terminator(C1-C 4) with : HindIII + BtgZI (C1-C4) , Expected bands : 3200+ 597bp ,Positive result: C1,C2

Cloned in alpha 1R

Fig.3:U=Uncut , C= Cut) Restriction digestion of AndersonJ23115:LacO -ECFP -Double Terminator (C1-C 4), with : BamHI (C1-C4) , Expected bands:2847 +952 bp. Positive result: C1,C2,C3,C4.

Experimental Use and Experinece

This part is used in BBa_K3505036

Extra Engineering Use

This part ,AndersonJ23114:LacO: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.

Fig.4: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.

We combined this part with BBa_K3505027 a LacI regulated by an inducible promoter of SCFAs. Adding SCFAs ,LacI is expressed and the transcription unit of ECFP is repressed.

We characterised the part BBa_K2924016, a SCFAs' inducible promoter Flic and concluded that the most optimal concentration of SCFAs is 2mM, so we simulated the proof of concept of our project adding 2mM of SCFAs , as shown below.

Fig.5:NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM acetate.

Fig.6:NOT-GATE-regulated ECFP fluorescence after in the presence or absence of 2mM propionate.

Sequence and Features