Difference between revisions of "Part:BBa K3610056"

Revision as of 01:25, 28 October 2020

FRB / SmallBit NanoLuc

This part encodes the SmallBit protein from the split-NanoLuc system fused to the FKBP–rapamycin binding (FRB) domain of the mammalian target of rapamycin (mTOR). This part can be used together with the Part:BBa_K3610054 to test the dimerization of the split-NanoLuc proteins via rapamycin induced dimerization of FKBP and FRB.

Characterization

We used this part to compare the performance of the codon optimized version of LargeBit with the original sequence in S. For a more detailed description of the experiment see Part:BBa K3610014

Plasmids containing the constructs FRB:SmallBit and FKBP12:LargeBit, were used to transfect <i>S. cerevisiae cells. This was done twice. In one case, the LargeBit sequence was codon optimized for expression in yeast (Part:BBa_K3610055), while one yeast culture was transfected with plasmids containing the original part (Part:BBa_K3610054). Both cell cultures contained a plasmid for expression of this part (Part:BBa_K3610056).

Samples from these cell cultures were examined with a luminometer of the type Synergy H1. Two types of treatments were performed for each sample, with and without addition of rapamycin. Each type of measurement was performed multiple times. For each experiment, the OD600 values of the samples were adjusted to the same value.

We conducted 2 luminescence assays.

Figure 1: Average Luminescence levels over time

Figure 2: Average Luminescence levels over time. Second measurement.

The results clearly demonstrate how rapamycin increases dimerization rates of the two NanoLuc parts. Suggesting that rapamycin induced protein-protein interaction with FRB and FKBP12 is possible for the NanoBit system.cerevisiae</i>.

Sequence and Features