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_K2736101 and BBa_K2736103, were created to be used as BRET pair to measure the activity of our customized Tar receptors (BBa_K2736106, BBa_K2736108). To test whether it was possible to switch ligand binding domains of the Tar receptor and two component receptors we created BBa_K2736105, and BBa_K2736109. During our project we proved the functionallity of the parts BBa_K2736101 and BBa_K2736105, which were submitted to the registery.

Table 1: Acquired Biobricks
NameBiobrick NumberSizeFunctionObtained
RFPBBa_K516032828Red fluorescenceIsolated
PromoterBBa_K60800356Strong promoter with medium ribosome binding siteIsolated
eYFPBBa_I15017724Yellow fluorescenceIsolated
CheZBBa_K629003645CheZ chemotaxis regulator protein phosphatase for CheYIsolated
Copper promoter-98Promoter responsive to phosphorylated CusR Ordered
Custom receptorBBa_K27361091505Fusion protein of CusS with the Tar ligand binding domain Ordered
1/5A::CheZ::RlucBBa_K27361031655CheZ fused to RlucOrdered
CheY-390Chemotaxis response regulator proteinOrdered
Table 2: Created Biobricks
NameBiobrick NumberSizeFunctionObtained
1/5A::eYFP::CheYBBa_K27361011865Chemotaxis responsive BRET pair (Part 1)Created
1/5A::CheZ::Rluc BBa_K27361031655Chemotaxis responsive BRET pair (Part 2)Created
Cu_prom::RFPBBa_K2736105930RFP behind a promoter that responds to CusRCreated
Tar(E491A)BBa_K27361061703Tar receptor protein with a E->A substitutionCreated