Successful Results1: Successful extraction lysis, and TEM imaging of exosomes:
We’ve used total exosome isolation kit (from medium) to extract the exosomes. The exosomes is visible in the bottom of the tube after centrifuge. After ripa lysis, SDS-PAGE results shown correct protein bands for exosomes. TEM result of paraformaldehyde fixed exosomes proves that exosomes extracted using this method has the correct shape and size.
2: CD63-Vhb Fusion Protein can transport Vhb into exosomes and the function of Vhb is not disturbed.
Cell content western blotting first proved successful translation of PNCV plasmids inside HEK 293T cells. Exosome content shown successful result of fusion protein band around 40 kda. TEM result of sample shown that the protein doesn’t effect the shape or the concentration of exosomes. COD result compared to normal exosomes shown that the fusion protein contains heme group that increases the chemical demand of exosomes and thus proving the function of Vhb is not disturbed. Also, color difference can be seen between normal and fusion-protein containing exosomes.
3: WW3-Vhb can be actively transported into exosomes, but more experiment is needed.
Cell content western blotting first proved successful translation of PNW3V plasmids inside HEK 293T cells. Exosome content shown pale fusion protein band around 30kda. TEM result of sample shown that the protein doesn’t effect the shape or the concentration of exosomes. COD result compared to normal exosomes shown that the COD value increased but the difference is not as huge as PNCV transfected once.
4:COD value can be used to test loading of Vhb into exosomes.
Repeat test on COD value of normal exosome shown that normal exosomes extracted from different plates have approximately the same base COD value. A difference can be seen between regular, PNV transfected and PNCV transfected exosomes. WW3 transfected does not show a significant difference.
5: Homologous Recombination+Linearized plasmid Can be Used in Part Submission and is both Fast and Effective.
Clone Express Kit from Vazyme is used in our parts submission process. As we can not get the restriction enzyme PstI due to time and cost, we’ve successfully used designed primer to create Homologous Region that can be recombined with linearized DNA provided by iGEM. Colony PCR and sequencing result both conformed the sequence of the parts. The process only needs two days and no restriction or ligation or gel experiment is needed.
Not So Successful Results1: WW4 expression failure
No bands of WW Tag4-Vhb is seen in western blotting either in cell or exosomes. Also, exosomes extracted from WW Tag4 transfected cells has irregular shape and contains a lot of unknown fragments, meaning that either the cell is contaminated or the plasmid is not constructed properly.2: Hemin do not contribute to the effectiveness of our designed exosomes.
We’ve added 3ug/ml hemin into the medium to see if hemin can contribute to the effectiveness of our designed exosomes. But after incubation, microscopic view shown that the hemin crystals are not dissolved in the medium. COD results also shown that no statistical difference can be seen between H+ and H- exosomes.3: The effectiveness of Ndfip1 hasn’t been tested
Due to the lack of samples, the effectiveness of ndfip1 on active loading of WW-Tagged Vhb can not be proven. Further research is needed.