Difference between revisions of "Part:BBa K2796028"
Line 16: Line 16:
 
<li>Here is the final goal that we designed for this project.</li></ul>
 
<li>Here is the final goal that we designed for this project.</li></ul>
 
[[File:2018 LZU-CHINA vesicle attacker.png|600px|thumb|center|]]
 
[[File:2018 LZU-CHINA vesicle attacker.png|600px|thumb|center|]]
 +
<ul>
 
It combines with exosome booster and miR attacker (miR135b-3p, miR-942-5p,miR768-5p) under control of induced promoter (hypoxia,tetracycline, biotin and galactose induced system). By adding different inducers into the cell culture, we can prompt engineered 293T cells to express different concentrations of miRNA. There are six combinations; then we can find the optimum combination of miRNA to achieve our goal——kill tumor cells.
 
It combines with exosome booster and miR attacker (miR135b-3p, miR-942-5p,miR768-5p) under control of induced promoter (hypoxia,tetracycline, biotin and galactose induced system). By adding different inducers into the cell culture, we can prompt engineered 293T cells to express different concentrations of miRNA. There are six combinations; then we can find the optimum combination of miRNA to achieve our goal——kill tumor cells.
 
More design details and description you can obtain from our wiki!
 
More design details and description you can obtain from our wiki!

Revision as of 06:53, 16 October 2018


Exosome booster

  • Considering that TIL cells can't secret enough exosomes under normal condition, we fuse three genes to increase the number of exosomes (Alenquer, 2015), thus increasing the transfer efficiency of exosomes.
  • Exosome booster refers to hSDC4-T2A-STEAP3-P2A-NadB.We identified STEAP3 (involved in exosomebiogenesis), syndecan-4(SDC4; supports budding of endosomal membranes to form multivesicular bodies), and a fragment of L-aspartate oxidase (NadB; possibly boosts cellular metabolism by tuning up the citric acid cycle) as potential synthetic exosome production boosters. Combined expression of these genes significantly increased exosome production.(Ryosuke Kojima et al. 2008)
T--LZU-CHINA--design1.png
    They combine with each other to boost exosomal transfer efficiency：
T--LZU-CHINA--design2.png
    In previous article, it use IRES (internal ribosome entry site,IRES) to connect three genes, we change it into linkers T2A and P2A to ensure proper expression of three genes.Then we use use reporter gene copGFP to manifest the expression of exosome booster with the promoter of 5-HRE-PminCMV.
  • Here is the final goal that we designed for this project.
2018 LZU-CHINA vesicle attacker.png
    It combines with exosome booster and miR attacker (miR135b-3p, miR-942-5p,miR768-5p) under control of induced promoter (hypoxia,tetracycline, biotin and galactose induced system). By adding different inducers into the cell culture, we can prompt engineered 293T cells to express different concentrations of miRNA. There are six combinations; then we can find the optimum combination of miRNA to achieve our goal——kill tumor cells. More design details and description you can obtain from our wiki!

    Modeling

    To increase the expression of exosomes, we added three enhanced genes to simulate the effect of increasing exosomes secretion through the model, providing guidance for our experiment. It includes three equations: a.transcriptional rate simulation of three booster genes:

    2018 LZU-CHINA model1.png

    b.Simulation of three booster genes transcriptional translation:

    2018 LZU-CHINA model2.png
    2018 LZU-CHINA model3.png

    a.the promotional concentrations of the three promotional genes for the secretion of exosomes:

    2018 LZU-CHINA model4.png

    Over time, the number of exosomes secreted by cells increased.

    b.Comparison between the expression levels of three promoter genes and those of non-promoter exosomes:

    2018 LZU-CHINA model5.png

    It demonstrates that exosome booster could dramatically increase exosome secrion. c.production rate of exosomes in cell:

    2018 LZU-CHINA model6.png

    This model predicts the production rate of exosome over time in cells.


    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 192
      Illegal BglII site found at 1777
    • 23
      COMPATIBLE WITH RFC[23]
    • 25
      INCOMPATIBLE WITH RFC[25]
      Illegal AgeI site found at 2657
      Illegal AgeI site found at 3277
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal SapI site found at 1367