Coding

Part:BBa_K1633008

Designed by: Chen Xi, Zhou Yu, Jiang Waner, Zhang Peng, Tian Chenfei   Group: iGEM15_NJU-China   (2015-09-13)

coding sequence to express nSMase 2 protein.

This part is a codon-optimized nSMase 2 gene CDS. When this part is inserted into pcDNA 3.1 vector and transfected into mammalian cells,it can express nSMase2 gene in mammalian cells and stimulate the secretion of exosomal siRNAs from cells.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 472
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1609
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 278
    Illegal BsaI.rc site found at 694
    Illegal BsaI.rc site found at 1600



USAGE AND BIOLOGY

Neutral sphingomyelinase 2 (nSMase 2) is a key regulatory enzyme generating ceramide from sphingomyelin and can actively induce exosome secretion from cells and trigger cellular export of small RNAs. The original sequence of nSMase 2 from Homo Sapiens is from NCBI. NCBI Gene ID is 55512. The sequence codon of this part was optimized. We ordered the sequence from a DNA synthesis company. When this part is inserted into pcDNA 3.1 vector and transfected into mammalian cells, it can express nSMase 2 gene in mammalian cells and stimulate the secretion of exosomal siRNAs from cells.

CHARACTERIZATION

Stimulation of exosome and exosomal siRNA secretion by introduction of nSMase 2

Extracellular vesicles (EVs) are generated through biogenetic mechanisms involving neutral sphingomyelinase (nsMase).

Because nSMase2 can stimulate both exosome production and siRNA loading to exosomes, we selected nSMase2 as a “molecular pump” to accelerate the amounts of exosomes released by cells and to promote the generation of exosomal siRNAs. A plasmid expressing nSMase2 was constructed and transfected into HEK293 cells to stimulate the secretion of exosomes and exosomal siRNA from HEK293 cells. As anticipated, both exosomes and exosomal siRNA secreted by HEK293 cells were increased after overexpressing nSMase2 in HEK293 cells.

NJU-China-parts-fig16.jpg

Figure 1. (A) Total amounts of exosomes (shown as total protein) secreted by HEK293 cells with or without the introduction of nSMase2. (B) Quantitative RT-PCR analysis of siRNA levels in exosomes secreted by HEK293 cells with or without the introduction of nSMase2.

We then performed nanoparticle tracking analysis (NTA) to have a more precise determination of the quantity and size of secreted exosomes. The use of Nanosight enabled quantification and size determination of the EV, as nanoparticles can be automatically tracked and sized based on Brownian motion and the diffusion coefficient. Because exosomes are more homogenous and generally smaller than most EVs with a diameter size ranging from 40 to 120 nm, the percentage of nanoparticles whose size ranges from 40 to 120 nm could be a good indicator of total amount of exosomes. The shift of peak of size distribution of EV from 170 nm to 120 nm and the significant raise of the peak indicated the increase of relative level of exosomes in secreted EVs after overexpression nSMase2 in HEK293 cells. From what we have discussed above, the improvement of manufacturing exosomes by overexpressing nSMase2 is proved to be feasible and effective.

NJU-China-parts-nsmase2.jpg


Figure 2. Characterization of secreted exosomes after overexpression of nSMase2 in HEK293 cells. (A and B) Representative screen shots of the NTA videos for EV from HEK293 cells under normal condition (left) or after transfection with nSMase2 plasmid (right). (C and D) Size and intensity of EV from HEK293 cells under normal condition (left) or after transfection with nSMase2 plasmid (right). (E) Concentration of different particle sizes of exosomes with (red line) or without (blue line) transfection with nSMase2 plasmid. The peak of size distribution of EV shifted from 170 nm to 120 nm after transfection with nsMase2 plasmid, indicating the increase in quantity of secreted exosomes.

Contribution: NJU-China 2020

nSMase2 was overexpressed in HEK293T cells, mRNA expression was detected by RT-QPCR and protein expression was detected by Western Blotting.

T--NJU-China--10.jpg

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.

T--NJU-China--11.jpg

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.

T--NJU-China--12.jpg

Figure5. total amounts of exosomes (shown as total protein) secreted by HEK293 cells with the introduction of nSMase2, KIBRA and hSDC4-STEAP3-NadB.

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