Composite

Part:BBa_K3258017

Designed by: Alec Lourenco   Group: iGEM19_Stanford   (2019-10-16)
Revision as of 13:02, 21 October 2019 by Apandit42 (Talk | contribs) (→‎Testing in In Vitro)


Self-amplifying fluorescent RNA (MDV-Q-beta-Spinach)

This is a part composite composed of self-encoded Qbeta replicase with a Spinach aptamer attached, both sequences flanked by midivariant regions recognized by Qbeta, referred to as MDV regions upstream and downstream. When expressed in E. coli cell free extract supplemented with DFHBI, Spinach fluorescence increases due to the replication of the RNA by the Qbeta replicase enzyme. This creates a self replicative cycle, and the amplification of the Qbeta and Spinach aptamer.

Testing in In Vitro

QBInVitroInitialTest.png

The functionality and operation of the MDV-Qbeta-Spinach construct was tested in Sigma 70 myTXTL Cell Free Cell Lysate. Initial tests were conducted comparing fluorescent output from another construct, mRFP-Spinach, with the fluorescent output of MDV-Qbeta-Spinach. Trials were conducted running reactions with each construct at the same time at 29 degrees Celsius for 18 hours and measuring fluorescent output. Results showed significant difference in fluorescent output between MDV-Qbeta-Spinach and mRFP-Spinach, suggesting that MDV-Qbeta-Spinach was operational in vitro in the used cell free lysate.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 180
    Illegal XbaI site found at 2231
    Illegal SpeI site found at 2107
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 146
    Illegal SpeI site found at 2107
    Illegal NotI site found at 2213
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 172
    Illegal BglII site found at 2228
    Illegal BamHI site found at 1538
    Illegal BamHI site found at 1681
    Illegal XhoI site found at 168
    Illegal XhoI site found at 1212
    Illegal XhoI site found at 2222
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 180
    Illegal XbaI site found at 2231
    Illegal SpeI site found at 2107
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 180
    Illegal XbaI site found at 2231
    Illegal SpeI site found at 2107
    Illegal NgoMIV site found at 2120
    Illegal NgoMIV site found at 2149
    Illegal AgeI site found at 1694
  • 1000
    COMPATIBLE WITH RFC[1000]


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