Difference between revisions of "Part:BBa K3247000"
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<partinfo>BBa_K324700 short</partinfo>
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<partinfo>BBa_K3247000 short</partinfo>
  
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Universal Bacterial Expression Resource (UBER) is a system containing T7 RNA Polymerase (T7 RNAP) which can be used for the expression of genes under T7 promoter. UBER includes T7 RNAP and TetR repressor which provides negative feedback on the polymerase levels to help reduce toxicity.  
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RNA thermometers are a form of translational regulation. Our RNA thermometers were designed to have a high fold change between 25°C and 30°C, which is a significantly lower temperature range than existing thermometers that are generally optimized for expression at 37°C or higher.
  
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===Design Notes===
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The stem-loop structure of the thermometer was created by taking the complement of the ribosome binding site (RBS) and adding nucleotides to either side. The additional bases were mutated; the resulting altered sequence is known as the variable region. Mutating the variable region keeps the RBS intact while modifying the RNA thermometer secondary structure to change the melting temperature. There is no internal BsaI cut site, since it would render the thermometer incompatible with our assembly method. The part is TypeIIS compatible. Figure 1a-c showing the RNA thermometer structures were designed using NUPACK software.
  
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===Usage and Biology===
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UBER consists of a TetR gene under control of a T7 promoter and a T7 RNA polymerase gene under the transcriptional control of a broad-host priming promoter as well as a TetR-repressible T7 promoter. The priming promoter starts transcription of the T7 RNAP, which then goes on to transcribe itself, creating a positive feedback loop in which increasing levels of T7 RNAP leads to increased transcription of the T7 RNAP gene. High levels of T7 RNAP may be toxic to cells; to prevent this toxicity, T7 RNAP also transcribes TetR, which binds to the TetR-repressible promoter and blocks transcription of T7 RNAP, creating negative feedback control to keep levels of T7 RNAP consistent. This pool of T7 RNAP can be used to transcribe any additional genes under a T7 promoter.  
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<img src="https://2019.igem.org/wiki/images/0/07/T--Rice--0125.png" width = 60%>
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This submitted UBER was adapted from the work of Kushwaha and Salis; the modifications made were: removal of yeast NLSs from the T7 RNAP and TetR genes, replacement of the uncharacterized priming promoter with a characterized broad-host priming promoter, and removal of an internal cut sequence.
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<p><b>Figure 1a.</b> Predicted RNA thermometer structure at 25 °C.</p>
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<img src="https://2019.igem.org/wiki/images/5/50/T--Rice--0130.png" width = 60%>
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<p><b>Figure 1b.</b> Predicted RNA thermometer structure at 30 °C.</p>
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<img src="https://2019.igem.org/wiki/images/0/06/T--Rice--0137.png" width = 60%>
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<p><b>Figure 1c.</b> Predicted RNA thermometer structure at 37 °C.</p>
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===Characterization===
 
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<p>The function of UBER containing our modification was compared with the original system from Kushwaha and Salis (Figures 1,2). <i>E. coli</i> DH10B </p> cells co-transformed with either original or modified UBER and an expression cassette containing mKate2 under T7 promoter were grown for 15 hours at 37 C in LB. Figures 1 and 2 show fluorescence time course for modified (Figure 1) and original (Figure 2) UBER. The results demonstrate that modifications introduced do not interfere with UBER function and result in higher fluorescence/OD compared to the original system.
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<img src="https://2019.igem.org/wiki/images/3/39/T--Rice--Nochill1.png" width = 60%>
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<p><b>Figure 2.</b> Fluorescence intensity at 25 °C, 30 °C, and 37 °C.</p>
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<partinfo>BBa_K3247000 SequenceAndFeatures</partinfo>
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<img src="https://2019.igem.org/wiki/images/3/39/T--Rice--Nochill1.png" width = 60%>
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===Source===
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<p><b>Figure 1</b>: Fluorescence intensity at 25°C, 30 °C, and 37 °C.</p>
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The thermometers were designed de novo using NUPACK and VSAlgorithm
  
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K2540011 SequenceAndFeatures</partinfo>
 
  
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===Functional Parameters===
 
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<partinfo>BBa_K2540011 parameters</partinfo>
 
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===References===
 
===References===
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J. N. Zadeh, C. D. Steenberg, J. S. Bois, B. R. Wolfe, M. B. Pierce, A. R. Khan, R. M. Dirks, N. A. Pierce. NUPACK: analysis and design of nucleic acid systems. J Comput Chem, 32:170–173, 2011. (pdf)
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Latest revision as of 23:29, 21 October 2019

RNA Thermometer NoChill-01

RNA thermometers are a form of translational regulation. Our RNA thermometers were designed to have a high fold change between 25°C and 30°C, which is a significantly lower temperature range than existing thermometers that are generally optimized for expression at 37°C or higher.

Design Notes

The stem-loop structure of the thermometer was created by taking the complement of the ribosome binding site (RBS) and adding nucleotides to either side. The additional bases were mutated; the resulting altered sequence is known as the variable region. Mutating the variable region keeps the RBS intact while modifying the RNA thermometer secondary structure to change the melting temperature. There is no internal BsaI cut site, since it would render the thermometer incompatible with our assembly method. The part is TypeIIS compatible. Figure 1a-c showing the RNA thermometer structures were designed using NUPACK software.

Figure 1a. Predicted RNA thermometer structure at 25 °C.

Figure 1b. Predicted RNA thermometer structure at 30 °C.

Figure 1c. Predicted RNA thermometer structure at 37 °C.

Characterization

Figure 2. Fluorescence intensity at 25 °C, 30 °C, and 37 °C.


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 7
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 7
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 7
  • 1000
    COMPATIBLE WITH RFC[1000]


Source

The thermometers were designed de novo using NUPACK and VSAlgorithm


References

J. N. Zadeh, C. D. Steenberg, J. S. Bois, B. R. Wolfe, M. B. Pierce, A. R. Khan, R. M. Dirks, N. A. Pierce. NUPACK: analysis and design of nucleic acid systems. J Comput Chem, 32:170–173, 2011. (pdf)