Difference between revisions of "Part:BBa K5301017"

Revision as of 07:37, 1 October 2024

sGFP1-10 tether is composed of mSA, a 3C linker, a 6His tag, and sGFP 1-10.

sGFP1-10 tether is composed of mSA, a 3C linker, a 6×His tag, and sGFP 1-10.We construct membrane protein dimers through the interaction between biotin and streptavidin, as well as the self-assembly mechanism of sGFP.

Usage and Biology

mSA is used for binding to biotinylated proteins, and sGFP1-10 and sGFP11 self-assemble to form GFP, thereby creating a protein dimer that can report the outcome with fluorescence.

Characterization

PCR

We conducted colony PCR validation to assess the feasibility of the plasmid pathway, as shown in Figure 1, where the corresponding bands are very clear, indicating that the target fragments were successfully introduced into the plasmid and transformed into the bacterial strain.

SSDS-PAGE

SDS-PAGE was used to verify the purification of the sGFP1-10 tether. Due to the particularity of mSA, we simultaneously purified the soluble proteins from the supernatant and the inclusion body proteins from the pellet after bacterial lysis. It can be observed that the concentration and purity of the purified inclusion body proteins are high, while the concentration of the soluble proteins is low. Further exploration of soluble protein expression or assessment of inclusion body protein activity can be conducted subsequently.

ELISA

Sequence and Features