Difference between revisions of "Part:BBa K3490010"

 
 
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The bacteria will continuously express TetR and GFP. After adding tetracycline into the culture, TetR will associate with tetracycline then binds on pTet which decreases the expression of GFP.
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Our first kill switch design uses TetR fused with VP16, tet-off system<sup>[1]</sup>to control lysozyme as our biosafety to prevent our engineered bacteria surviving when the contact lens accidently breaks (BBa_K3490011). (Fig.1)
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<img src="https://static.igem.org/mediawiki/parts/a/ad/T--NCKU_Tainan--BBa_K3490011.png" style="width:35%;">
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<br>Fig. 1 Our design about the killswitch using tet system
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After trying many times to transform our IDT synthesis into <i>E. coli</i>, we failed as precedent because VP16 hasn’t been applied to prokaryotic. To prove the concept that bacteria will die when escaping the contact lens, we found the plasmid from iGEM kit BBa_K611059, which has <i>pTet</i> promoter with GFP, and pBAD promoter with <i>TetR</i>. We planned to use AnhydroTetracycline to do the GFP express experiment<sup>[2]</sup>, but it is too expensive to do it, so we found another way to prove our ideas. When arabinose and heat inactivate chlortetracycline (CTC) [2] or Tetracycline (Tc) is added into the liquid culture, it should express a different amount of GFP, as to proof heat inactivate chlortetracycline or Tetracycline is crucial to the kill switch system.
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<br>Still in progress......
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 15:03, 24 October 2020


Verifying the function of tetR system.

Our first kill switch design uses TetR fused with VP16, tet-off system[1]to control lysozyme as our biosafety to prevent our engineered bacteria surviving when the contact lens accidently breaks (BBa_K3490011). (Fig.1)


Fig. 1 Our design about the killswitch using tet system

After trying many times to transform our IDT synthesis into E. coli, we failed as precedent because VP16 hasn’t been applied to prokaryotic. To prove the concept that bacteria will die when escaping the contact lens, we found the plasmid from iGEM kit BBa_K611059, which has pTet promoter with GFP, and pBAD promoter with TetR. We planned to use AnhydroTetracycline to do the GFP express experiment[2], but it is too expensive to do it, so we found another way to prove our ideas. When arabinose and heat inactivate chlortetracycline (CTC) [2] or Tetracycline (Tc) is added into the liquid culture, it should express a different amount of GFP, as to proof heat inactivate chlortetracycline or Tetracycline is crucial to the kill switch system.
Still in progress......



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
    Illegal NheI site found at 755
    Illegal NheI site found at 1068
    Illegal NheI site found at 1091
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]