Engineering success for SHSBNU_China

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.

 4XT: BBa_K4672000

To do this, we read the published paper and notice 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 as part BBa_K4672000 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.

 BBa_K4672001

After the monomer expression of 4XT , we designed a new plasmid to realize polymerize, in which we fused the C- and N-terminal of a fast-reacting SI pair to 4XT, named IntC-4XT-IntN (Part number BBa_K4672001). According to the published papers, SIs catalyze splicing reactions spontaneously, which lead to the covalent link between fusion partners through peptide bonds.

Protocol we use:

We asked the biological company 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.

 BBa_K4672002

Besides of the monomer part 4XT and polymer part IntC-4XT-IntN, we also constructed pET28a(+)-IntC-8XT-IntN plasmid in which the subunit consisting of eight Ig domains to see whether more subunit could help with better polymerization

Protocol we use:

We asked the biological company 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.