Team:Utrecht/Parts

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Several biobricks from the distribution kit were used during our project. These were retrieved from the kit using the protocols provided by iGEM, transformed into E. coli and isolated in elution buffer NE. We also ordered several constructs from IDT (Table 1). These Biobricks were used to create several new biobricks that were relevant to our project. BBa_K2736000 and BBa_K2736001, were created to be used as BRET pair to measure the activity of our customized Tar receptors (BBa_K2736003, BBa_K2736004, and BBa_K2736005). To test whether it was possible to switch ligand binding domains of the Tar receptor and two component receptors we created BBa_K2736002, and BBa_K2736006.

Table 1: Used Biobricks

Name	Biobrick Number	Size	Size	Obtained
RFP	BBa_K516032	828	Red fluorescence	isolated
Promoter	BBa_K608003	56	Strong promoter with medium ribosome binding site	isolated
eYFP	BBa_I15017	724	Yellow fluorescence	isolated
CheZ	BBa_K629003	645	CheZ chemotaxis regulator protein phosphatase for CheY	isolated
Copper promoter	-	98	Promoter responsive to phosphorylated CusR	ordered
Custom receptor	BBa_K2736006	1505	Fusion protein of CusS with the Tar ligand binding domain	ordered
1/5A::CheZ::Rluc	BBa_K2736002	1655	CheZ fused to Rluc	ordered
CheY	-	390	Chemotaxis response regulator protein	ordered

Table 2: Created Biobricks

Name	Biobrick Number	Size	Size	Obtained
1/5A::eYFP::CheY	BBa_K2736000	1865	Chemotaxis responsive BRET pair	Created
1/5A::CheZ::Rluc	BBa_K2736001	930	RFP behind a promoter that responds to CusR	Created
Cu_prom::RFP	BBa_K2736002	1655	Chemotaxis responsive BRET pair	Created
Tar(E491A)	BBa_K2736003	1703	Tar receptor protein with a E->A substitution	Created
Tar(Q309A)	BBa_K2736004	1703	Tar receptor protein with a Q->A substitution Created
Tar(E491A, Q309A)	BBa_K2736005	1703	Tar receptor with two amino acid mutations Created