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Under the combination of P70b promoter and UTR2, green fluorescent protein can be expressed.
 
Under the combination of P70b promoter and UTR2, green fluorescent protein can be expressed.
  
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===Usage and Biology===
 
===Usage and Biology===
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According to the lecture, we selected three promoters for Cell-Free systems: P70a, P70b and P70c and combined with three Ribosome Binding Sites (RBS) UTR1, UTR2 and UTR3 to express deGFP gene. The fluorescence intensity of deGFP was measured by adding varied concentrations of plasmids into the Cell-Free system. Simultaneously, experimental results and modeling data were compared, and the optimal promoter RBS combination was obtained in order to express deGFP in a Cell-Free.
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Result
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<p style="text-align: center:"> Figure 1. Comparing expression in TXTL reactions to the model. A) Maximum rate of deGFP synthesis expressed from varying concentrations of P70b-UTR2-deGFP. B,C,D) Kinetics of deGFP synthesis for the plasmid P70b-UTR2-deGFP at three concentrations: one below, one at, and one above saturation  (A) 1 nM, (B) 5 nM, (C) 10 nM, shown for the first 3 hours.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 13:59, 19 October 2021


P70b-UTR2-deGFP

Under the combination of P70b promoter and UTR2, green fluorescent protein can be expressed.

Usage and Biology

According to the lecture, we selected three promoters for Cell-Free systems: P70a, P70b and P70c and combined with three Ribosome Binding Sites (RBS) UTR1, UTR2 and UTR3 to express deGFP gene. The fluorescence intensity of deGFP was measured by adding varied concentrations of plasmids into the Cell-Free system. Simultaneously, experimental results and modeling data were compared, and the optimal promoter RBS combination was obtained in order to express deGFP in a Cell-Free.

Result

Figure 1. Comparing expression in TXTL reactions to the model. A) Maximum rate of deGFP synthesis expressed from varying concentrations of P70b-UTR2-deGFP. B,C,D) Kinetics of deGFP synthesis for the plasmid P70b-UTR2-deGFP at three concentrations: one below, one at, and one above saturation (A) 1 nM, (B) 5 nM, (C) 10 nM, shown for the first 3 hours.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 70
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]