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.
Learning upon successful synthesis of 4XT, we proceeded to design two other parts, constructed and tested them with proper experiment conditions for polymerized expression. These parts are documented as IntC-4XT-IntN (BBa_K4672001) and IntC-8XT-IntN (BBa_K4672002)
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 - Characterization
Team: SHSBNU-China
Protein Expression
The development of synthetic biology has solved many problems for mankind, one of which is to provide high-performance polymers. Not only do the biosynthesized polymers exhibit higher strength, toughness and better damping energy, they also avoid from producing toxic byproducts in the whole process. This year, SHSBNU_China concentrated on improving the performance of artificial heart valves with megadalton titin polymers produced by E.coli. We believe our project will expand the connection between synthetic biology and biomedical field.
We searched the published papers and noticed that titin is a muscle protein, in which the most essential part is hundreds of folded immunoglobulin (Ig) domains in repetitive sequence. Following the paper, we chose to polymerize a relatively rigid subunit consisting of four Ig domains from the I-band of the rabbit soleus muscle titin, which has been uploaded in this part and named as 4XT.
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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