Part:BBa_K4672000
4XT
Titin is a muscle protein, in which the most essential part comprises of hundreds of folded immunoglobulin (Ig) domains in a repetitive sequence. Following the previous research work, we chose to polymerize a relatively rigid sub-unit consisting of four Ig domains from the I-band of the rabbit soleus muscle titin, which is coded by the sequence on this page and named as 4XT.
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 560
iGEM 2023 SHSBNU - Contribution
Team: SHSBNU-China
Production of NC-2Rep in Supernatant
Protocol we use: We asked the biological company to synthesize 4XT 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. As shown in the figure, both the two environment showed the expression bands of pET28a(+)-4XT, which only appeared in the group with IPTG. The molecular weight of this band was in line with the expectation at around 43 kDa. Besides, the expression for 20 h under 16℃ had better performance. The above results confirmed our success in titin monomer expression, which preferred a lower temperature and longer induction time.
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