Difference between revisions of "Part:BBa K4586027"

Revision as of 14:08, 12 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.

(2)Booster gene 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.

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.

Experimental Characterization

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 lane (P3) including NAdB, 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 in lane (P2) and (P3).

After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure. This plasmid contains 1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64)) 2-Booster gene 1 (SDC4, STEAP3) 3-Booster gene 2 (NAdB)

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 614
Illegal BglII site found at 1152
Illegal BglII site found at 2743
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 3623
Illegal AgeI site found at 4243
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 2333

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+After the ligation step, we did culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid.
+The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure.
+This plasmid contains
+-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64))
+-Booster gene 1 (SDC4, STEAP3)
+-Booster gene 2 (NAdB)
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