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Our Best Composite Part:BBa_K2557010

We combined three promoters, miniCMV promoter, EF1α promoter and Ubc promoter, with three recombinases, Bxb1, PhiC31 and TP901, and identified nine combinations. In the end, we chose TetO-miniCMV promoter-Bxb1 as our best composite part.

Function In Our Genetic Circuit

This part can convert the input signal of the upstream TEV into the output signal of the downstream RFP. When TEV is detached from the cell membrane, it functions as a protease, and the inhibition of the promoter by TetR is released, and the expression of Bxb1 recombinase is turned on. Finally, Bxb1 functions as a recombinase to initiate expression of RFP.

Characterization

TetR can effectively repress Tet operator in HEK 293T

Fig. 1 Inhibition of TetR on promoter with TetO sequence
(a)Fluorescence microscope observation of HEK 293T only transfected with plasmids containing promoters with TetO sequence.
(b)Fluorescence microscope observation of HEK 293T transfected with plasmids containing promoters with TetO sequence and tetR.

Bxb1 works well in HEK 293T

Fig. 1a Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration.
Fig. 1b The reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration.

Combining these data and, to our knowledge, we first constructed a TetO-regulated recombinase genetic circuit in mammalian cells, so we present BBa_K2557010 as the Our Best Composite Part.

Fig. 1 Bxb1 structure homology-model provided by SWISS-MODEL.

Composite Part List

Name	Type	Description	Length
BBa_K2557010	Composite	TetO-miniCMV promoter-Bxb1	1890
BBa_K2557002	Composite	Bxb1 attB-RFP-Bxb1 attP	935
BBa_K2557005	Composite	PhiC31 attB-mCherry-PhiC31 attP	907
BBa_K2557008	Composite	TP901 attB-mCherry-TP901 attP	949
BBa_K2557011	Composite	TetO-EF1α promoter-Bxb1	2070
BBa_K2557012	Composite	TetO-Ubc promoter-Bxb1	2258
BBa_K2557013	Composite	TetO-miniCMV-PhiC31	2212
BBa_K2557014	Composite	TetO-EF1α-PhiC31	2386
BBa_K2557015	Composite	TetO-Ubc promoter-PhiC31	2574
BBa_K2557016	Composite	TetO-miniCMV promoter-TP901	1935
BBa_K2557017	Composite	TetO-EF1α promoter-TP901	2109
BBa_K2557018	Composite	TetO-Ubc promoter-TP901	2297
BBa_K2557019	Composite	miniCMV promoter-Bxb1-RDF	2391
BBa_K2557020	Composite	EF1α promoter-Bxb1-RDF	2565
BBa_K2557021	Composite	Ubc promoter-Bxb1-RDF	2753
BBa_K2557022	Composite	miniCMV promoter-PhiC31-RDF	2676
BBa_K2557023	Composite	EF1α promoter-PhiC31-RDF	2850
BBa_K2557024	Composite	Ubc promoter--PhiC31-RDF	3038
BBa_K2557025	Composite	miniCMV promoter-TP901-RDF	1842
BBa_K2557026	Composite	EF1α promoter--TP901-RDF	2016
BBa_K2557027	Composite	Ubc promoter--TP901-RDF	2204
BBa_K2557028	Composite	TetO-miniCMV promoter- EGFP	1063
BBa_K2557029	Composite	TetO-EF1α promoter -EGFP	1237
BBa_K2557030	Composite	TetO-Ubc promoter- EGFP	1425
BBa_K2557031	Composite	miniCMV promoter-Anti-GFP-mnotch-TEV protease-NLS	2455
BBa_K2557051	Composite	EF1α promoter-TetR	1871