Part:BBa_K2619001
syndecan 4/SDC4/SYND4, syndecan-4/syntenin pathway
SDC4 is involved in syndecan-4/syntenin pathway. The formation of exosomes is controled by syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin, and it is due to interaction of sydecan`syntenin complex with Alix, an ESCRT (endosomal-sorting complex required for transport) III-binding protein. Syndecan-4 supports budding of endosomal membranes to form multivesicular bodies by interacts direactly with ALIX through LYPX(n)L motifs (Baietti et al., 2012). The activation Rac1 and RhoG requires interactions of syndecan-4 and its cytoplasmic partners synectin and syntenin, and this activation will further regulate exosome biosynthesis. therefore we used this gene to increase HEK293T's exosome biogenesis speed.
Literature Characterization by AFCM-Egypt
The study created a reporter construct by joining the C-terminus of CD63, one of the most used exosome markers, to nanoluc (nluc), a tiny and potent bioluminescence reporter10. After progressive centrifugation to eliminate masking signals12, luminescence in the cell-culture supernatant was measured. This reporter gene was co-transfected with plasmids expressing potential candidates for exosome production augmentation.
The study found STEAP3 syndecan-4 (SDC4), and (NadB) as potential synthetic exosome production boosters. Combined expression of these genes significantly increased exosome production, and a tricistronic plasmid vector ( known as exosome production booster), which guarantees that transfected cells receive all boosted genes at a fixed ratio ,produced a 15-fold to 40-fold increase (depending on cell conditions) in the luminescence signal in the supernatant.
Characterization By Mutational Landscape by AFCM-Egypt
In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (M169V) shows the highest epistatic fitness, while the lowest score was associated with the mutation (T144N).
Figure . An illustration of the effects of different mutations on the Epistatic Fitness of sdc4.
charactrization by mathematical modeling by AFCM-Egypt
Presence of SDC4 as a booster gene has a role in increasing the concentration of the produced engineered exosomes so it plays an effective role to increase the efficacy of the therapeutic agent.
We compared both condition of exosomes production when using booster genes and without it
(1)No booster genes with conditioned release
This Represents the relation between the activation of the internal domain of the Syn-Notch (represented as red line) and production of exosomes with specific cargo (represented as blue line) as the production of the engineered exosomes is initiated once the internal domain is activated.
(2)Booster gene with conditioned release
This Represents the relation between the activation of the internal domain of the Syn-Notch (represented as red line) and production of exosomes with specific cargo (represented as blue line) as the production of the engineered exosomes is initiated once the internal domain is activated.
Experimental Characterization by AFCM-Egypt
In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P2) including STEAP3 and SDC4, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P2).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 201
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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