Part:BBa_K4672001
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 701
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