Difference between revisions of "Part:BBa K4586010"

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lang=EN style='font-size:11.0pt;line-height:115%'>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.
 
lang=EN style='font-size:11.0pt;line-height:115%'>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.
 
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==Characterization By Mutational Landscape==
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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).
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
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lang=EN style='font-size:11.0pt;line-height:115%'>Figure . An illustration of the effects of different mutations on the Epistatic Fitness of sdc4.
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==References==
 
==References==
 
Kojima, R., Bojar, D., Rizzi, G., Hamri, G. C. E., El-Baba, M. D., Saxena, P., ... & Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nature communications, 9(1), 1305.
 
Kojima, R., Bojar, D., Rizzi, G., Hamri, G. C. E., El-Baba, M. D., Saxena, P., ... & Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nature communications, 9(1), 1305.

Revision as of 16:50, 3 October 2023


SDC4

Part Description

This part is an activator of signaling pathways to control cellular processes that arbitrate cell migration, proliferation, endocytosis, and mechano-transduction, as well as its ability to independently activate signaling pathways in response the target-ligand binding as well.

Usage

This part acts to strengthen the duration and intensity of downstream signaling as target-ligand binding occurs. We implemented this in our system to improve the efficacy of our therapeutic agent by increasing the default level of exosome synthesis within our engineered MSC as shown in figure 1.

Figure 1: This figure illustrates the design of our biological circuit coding for booster genes(SDC4,STEAP3 and NadB) and their role in increasing the synthetic capacity of MSCs to secrete exosomes that carry our therapeutic agent represented in Cas12k/gBAFF-R

Literature Characterization

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

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.

References

Kojima, R., Bojar, D., Rizzi, G., Hamri, G. C. E., El-Baba, M. D., Saxena, P., ... & Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson’s disease treatment. Nature communications, 9(1), 1305. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 201
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]