Part:BBa_K4672002
IntC-8XT-IntN
Besides of the monomer part 4XT (BBa_K4672000) and polymer part IntC-4XT-IntN (BBa_K4672001), we also constructed pET28a(+)-IntC-8XT-IntN plasmid in which the sub-unit consisting of eight (8X) Ig domains to see whether more sub-units could help with better polymerization and expression.
iGEM 2023 SHSBNU - Characterization
Team: SHSBNU-China
Protein Expression
Protocol for experiments:
We asked the biological company BGI to synthesize IntC-8XT-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.
However, this result is not ideal. We further asked for help and underwent a Western blot assay to see whether this is because of the low expression level, but western blot does not present bands either. We feel confused about the result and will fix it later.
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
Illegal SapI site found at 1859
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