Difference between revisions of "Part:BBa K4586027"

Revision as of 16:57, 8 October 2023

Booster genes (SDC4, STEAP3, NadB)

Usage

This composite part designed to increase the default number of exosomes secreted from 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.

charactrization by mathematical modeling

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-noth (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.

This Represents the relation between the activation of the internal domain of the syn-noth (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.

Improvement by our team

This composite part is an improvement for (BBa_K2796028) Designed by (iGEM18_LZU-CHINA).We developed this new approach to control the expression of these booster genes (SDC4, STEAP3, NadB) that enhance the exosomes secretion within our engineered mesenchymal stem cell, as we found out that constitutive overexpression of these genes may carry out multiple side effect in addition to depleting MSCs resources and impairing its function and survival, so we implemented the Cre loxP system for conditional gene expression to induce the expression of these booster genes by deleting (STOP) sequence that prevents the transcription of the downstream genes. Thus, the expression of booster genes as condition in the presence of Cre recombinase enzyme after vimentin syn notch activation.

This figure illustrates the conditional expression of our enhanced booster genes that increase the exosomal secretion.

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