This year the NJU-China team designed a new part KIBRA (BBa_K3335000), which promotes exosome release, and compared it with previous components CMV-nSMase2 (BBa_K1633008) and CMV-hSDC4-STEAP3-NadB (BBa_K2796028). We improved the expression of the original components at the mRNA and protein levels, filling in the gaps in the relevant parts. Our experiments showed that under the same transfection efficiency, HSDC4-Steap3-NADB could express more proteins better and promote exosome release better than nSMase2. And perfected the relevant information. A reference for future iGEM teams in the selection part process.
At the same time, we helped the SHTech iGEM team complete 3D printing related operations.
New data collected from laboratory experiments
We made some contributions to the correct expression of CMV-nSMase2, and characterized the expression of nSMase2 in HEK293T cells by QRT-PCR.
nSMase2 was overexpressed in HEK293T cells, mRNA expression was detected by RT-QPCR and protein expression was detected by Western Blotting.
Figure3.(A) nSMase2 mRNA relative expression in HEK293T cell (vs GADPH)
(B) nSMase2 protein relative expression in HEK29T cell (vs GAPDH)
From our experiments, we can see that nSMase2 is successfully overexpressed in HEK293T cells, which proves that we have indeed constructed an overexpressed cell line.
Next, we designed two other parts (CMV-KIBRA and CMV-hSDC4-STEAP3-NadB) to promote exosome release. We detected both mRNA levels and KIBRA protein expression.Because CMV-HSDC4-Steap3-NADB has been a very common element to promote exosome release, we only performed RT-qPCR to detect mRNA expression.
Figure4.(A) KIBRA mRNA relative expression in HEK293T cell (vs GADPH)
(B) NadB mRNA relative expression in HEK293T cell (vs GADPH)
(C) KIBRA relative expression in HEK293T cell (vs GADPH)
After confirming the normal expression of the three components, we attempted to compare the exosomes produced by the three parts. The results showed that KIBRA produced higher exosomes than the other two components.
Figure5. total amounts of exosomes (shown as total protein) secreted by HEK293 cells with the introduction of nSMase2, KIBRA and hSDC4-STEAP3-NadB.
In a word, we have characterized the original 2 parts. At the same time, according to literature and experimental results, we found that the new Part CMV-KIBRA designed by us could better promote exosome release, which helped us to choose exosome promoter in the future and also made contributions to other teams.