Difference between revisions of "Part:BBa K4672001"

Latest revision as of 04:03, 10 October 2023

IntC-4XT-IntN

We designed a new part to realize polymerization of 4XT BBa_K4672000, in which we fused the C- and N-terminal of a fast-reacting SI pair to 4XT, named IntC-4XT-IntN. SIs catalyze splicing reactions spontaneously, which lead to the covalent link between fusion partners through peptide bonds to polymerize into long fibrous threads.

iGEM 2023 SHSBNU - Characterization

Team: SHSBNU-China

Protein Expression

Protocol we use: We asked the biological company BGI to synthesize IntC-4XT-IntN sequence at first, the sequence was then constructed onto pET28a(+) plasmids, which is proper for protein purification and expression.

We further transformed the plasmids into E. coli BL21(DE3) strains.

The colony was grown up on the plate, and we picked a single colony using a sterile tip.

The single colony and tip were added it into 4 ml LB medium with the kanamycin and then cultured overnight.

When the bacteria solution reached OD600=0.6, we added 0.5 mM IPTG for induction and shook at 16℃ for 20 h or 37℃ for 4 h. We also set a control group and didn’t add IPTG into it.

After the expression was completed, we centrifuged the bacterial solution at 12000 rpm, discarded the supernatant, and then used RIPA as a lysis buffer to resuspend the bacteria.

For the cell lysate, we added loading buffer to heat at 96℃ for 10 min.

Finally we underwent SDS-PAGE assay and Coomassie brilliant blue staining for expression test.

The pET28a(+)-IntC-4XT-IntN worked well, as we observed cells produced a cluster of ultra-high molecular weight (UHMW) products up to and above 400 kDa in the top. Consistently with the former result, expression for 20 h under 16℃ had better performance. Taken together, we successfully produced titin polymer and found a better expression condition for UHMW production.

Sequence and Features