Difference between revisions of "Part:BBa K3335000"
 
(13 intermediate revisions by the same user not shown)
Line 11: Line 11:
 
<!-- Add more about the biology of this part here -->
 
<!-- Add more about the biology of this part here -->
 
===Usage and Biology===
 
===Usage and Biology===
−
 
−
<!-- -->
 
−
<span class='h3bb'>Sequence and Features</span>
 
−
<partinfo>BBa_K3335000 SequenceAndFeatures</partinfo>
 
−
 
−
 
 
<!-- Uncomment this to enable Functional Parameter display -->
 
<!-- Uncomment this to enable Functional Parameter display -->
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3335000 parameters</partinfo>
 
<partinfo>BBa_K3335000 parameters</partinfo>
 
<!-- -->
 
<!-- -->
−
 
 
==Contribution: NJU-China 2020==
 
==Contribution: NJU-China 2020==
−
<p>This year we want to use liver cells as a concept to treat lung cancer by overexpressing exosomes containing siRNA.</p>
+
<p>As previously reported, CMV-hSDC4-STEAP3-NadB can strongly promote exosome release. To develop the more efficient booster part for our strategy, we designed another two parts (CMV-KIBRA and CMV-nSMase2) to find out the most efficient design for our project. We tested all the three designs <i>in vitro</i>. The expression of each mRNA was confirmed by RT-qPCR after transfected to HEK293T cells.</p>
−
<p>In our interview with the professor, we learned that exosome production is likely to be inefficient, so we found the KIBRA protein.KIBRA as an adaptor - like protein that stabilizes Rab27a, which in turn controls exosome secretion.</p>
+
[[File:T--NJU-China--50001.png|500px|thumb|center|Figure 1. KIBRA, NadB, nSMase2 mRNA relative expression in HEK293T cell (vs GADPH)]]
−
 
+
<p>To further verify the expression of KIBRA and nSMase2 at protein level, we used Western Blotting experiment to prove that KIBRA and nSMase2 was indeed overexpressed in HEK293T cells.</p>
−
<p>[[File:T--NJU-China--1.png|800px|thumb|center|Figure 1）qRT-PCR analysis of plasmid-borne ppk1 expression in CPP ]] </p>
+
[[File:T--NJU-China--50002.png|400px|thumb|center|Figure 2. (A)Western Blotting result shows that nSMase2 is overexpressed in HEK293T cell.
 +
(B) Western Blotting result shows that KIBRA is overexpressed in HEK293T cell.]]
 +
[[File:T--NJU-China--50003.png|400px|thumb|center|Figure 3. Total amounts of exosomes (shown as total protein) secreted by HEK293 cells with the introduction of nSMase2, KIBRA and hSDC4-STEAP3-NadB.]]
 +
<p>After confirming the correct expression of these three genes, we further compared their ability to promote exosome secretion. To simplify the process, we first detected the total concentration of the exosomes produced by cells transfected each part. The result indicated that overexpression of KIBRA generates the highest amount of exosomes among these three groups. So we chose KIBRA as the candidate for further characterization.</p>
 +
<!-- -->
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K3335000 SequenceAndFeatures</partinfo>

Latest revision as of 10:23, 21 December 2020


We use KIBRA to control exosome secretion

KIBRA as an adaptor-like protein that stabilizes Rab27a, which in turn controls exosome secretion. Rab27a is stabilized by interacting with KIBRA, which prevents ubiquitination and degradation via the ubiquitin-proteasome pathway. KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a[1].

In our experiment, we used KIBRA to promote cell release of exosomes containing siRNA, thus increasing the efficiency of cell production and release of exosomes and improving the killing of tumor cells.

[1]Lin Song et al,.KIBRA controls exosome secretion via inhibiting the proteasomal degradation of Rab27a.Nature Communication,2019.

Usage and Biology

Functional Parameters

Contribution: NJU-China 2020

As previously reported, CMV-hSDC4-STEAP3-NadB can strongly promote exosome release. To develop the more efficient booster part for our strategy, we designed another two parts (CMV-KIBRA and CMV-nSMase2) to find out the most efficient design for our project. We tested all the three designs in vitro. The expression of each mRNA was confirmed by RT-qPCR after transfected to HEK293T cells.

Figure 1. KIBRA, NadB, nSMase2 mRNA relative expression in HEK293T cell (vs GADPH)

To further verify the expression of KIBRA and nSMase2 at protein level, we used Western Blotting experiment to prove that KIBRA and nSMase2 was indeed overexpressed in HEK293T cells.

Figure 2. (A)Western Blotting result shows that nSMase2 is overexpressed in HEK293T cell. (B) Western Blotting result shows that KIBRA is overexpressed in HEK293T cell.
Figure 3. Total amounts of exosomes (shown as total protein) secreted by HEK293 cells with the introduction of nSMase2, KIBRA and hSDC4-STEAP3-NadB.

After confirming the correct expression of these three genes, we further compared their ability to promote exosome secretion. To simplify the process, we first detected the total concentration of the exosomes produced by cells transfected each part. The result indicated that overexpression of KIBRA generates the highest amount of exosomes among these three groups. So we chose KIBRA as the candidate for further characterization.

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 335
    Illegal BglII site found at 2999
    Illegal BamHI site found at 246
    Illegal BamHI site found at 442
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 435
    Illegal BsaI site found at 2598
    Illegal BsaI.rc site found at 1920
    Illegal BsaI.rc site found at 2644