Part:BBa_K2796028

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)

They combine with each other to boost exosomal transfer efficiency：

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.

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:

• transcriptional rate simulation of three booster genes
  
• simulation of three booster genes transcriptional translation:
  

  The interpretation of the parameters is shown in the following table
  

  

  From the formula, we can obtain three model about exosome booster.
  a.the promotional concentrations of the three promotional genes for the secretion of exosomes:

  

  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:

  

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

  

  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