Difference between revisions of "Part:BBa K2918040"

 
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<partinfo>BBa_K2918040 short</partinfo>
 
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Genetic implementation of an incoherent feed forward loop (iFFL) in which a stabilized 0.1 T7 promoter is controlling GFP expression.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2918040 SequenceAndFeatures</partinfo>
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The two transcriptional units in this composite part are oriented outwards.
  
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===Usage and Biology===
 
===Usage and Biology===
  
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A Incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal. This is achieved by the introduction of a repressor.
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<div><ul>
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<center>
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  <li style="display: inline-block;"> [[File:T--TUDelft--ifflparts.png|thumb|none|550px|<b>Figure 1:</b> Overview of incoherent feed-forward loop]] </li>
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</center>
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    </ul></div>
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iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, <html><a href="https://parts.igem.org/Part:BBa_K2918008/">TALE </a></html> proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code <html><a href="#Doyle2013">(Doyle, Stoddard et al., 2013)</a></html>. The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (<html><a href="https://parts.igem.org/Part:BBa_K2918009/">0.1 T7sp1 promoter</a></html>, <html><a href="https://parts.igem.org/Part:BBa_K2918010">0.5 T7sp1 promoter</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918011">P<sub>BHR</sub>sp1 promoter</a></html>). <br> In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP.
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</figure>
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    <br>
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    <figure>
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      <img src="https://2019.igem.org/wiki/images/8/82/T--TUDelft--TALEanimation.gif" style="width:100%;border:1px solid #00a6d6;"  alt="iFFL" >
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      <figcaption ><br><b>Figure 2: Animation of TALE protein binding to the promoter of a GOI.</b> The binding of the TALE protein represses the expression of a GOI. </figcaption>
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    </figure>
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</html>
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When transcriptional units are placed in series, read through transcription due to low efficiency of terminators can occur. This could influence the behavior of the genetic circuit. Hence, the transcriptional units in the circuit are oriented to achieve insulation from influence of neighboring transcriptional unit.
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An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. iFFL based on broad host range promoters (<html><a href="https://parts.igem.org/Part:BBa_K2918000/">P<sub>BHR </sub>promoter</a></html> and <html><a href="https://parts.igem.org/Part:BBa_K2918011">P<sub>BHR</sub>sp1 promoter</a></html>)  has been demonstrated below to achieve controllable expression between <i> E.coli </i> and <i> P.putida </i>.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2918040 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2918040 SequenceAndFeatures</partinfo>

Revision as of 11:27, 19 October 2019


T7 promoter based optimized iFFL

Genetic implementation of an incoherent feed forward loop (iFFL) in which a stabilized 0.1 T7 promoter is controlling GFP expression.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 250
    Illegal XhoI site found at 3335
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
    Illegal AgeI site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]

The two transcriptional units in this composite part are oriented outwards.

Usage and Biology

A Incoherent feed-forward loop (iFFL) is a unique control systems motif where the output signal is robust to changes in the input signal. This is achieved by the introduction of a repressor.

  • Figure 1: Overview of incoherent feed-forward loop

iFFL can be applied to genetic circuits to achieve expression independent from copy number, transcriptional and translational rates. To implement the iFFL in a genetic circuit, TALE proteins can be used. These proteins consist of repeats where 12th and 13th amino acids can vary, these are called the repeat variable diresidue (RVD). RVDs have been shown to bind to DNA in a simple one-to-one binding code (Doyle, Stoddard et al., 2013). The direct correspondence between amino acids allows scientists to engineer these repeat regions to target any sequence they want. In our system, we used the TALE protein as a repressor by engineering promoters to contain the binding site of this specific TALE protein (0.1 T7sp1 promoter, 0.5 T7sp1 promoter and PBHRsp1 promoter).
In our genetic circuit, a unrepressed promoter controls the expression of TALE protein while the promoters with the TALE binding sites drive expression of GFP.

iFFL

Figure 2: Animation of TALE protein binding to the promoter of a GOI. The binding of the TALE protein represses the expression of a GOI.

When transcriptional units are placed in series, read through transcription due to low efficiency of terminators can occur. This could influence the behavior of the genetic circuit. Hence, the transcriptional units in the circuit are oriented to achieve insulation from influence of neighboring transcriptional unit.

An interesting application of the iFFL is to achieve controllable gene expression across different bacterial species. Gene expression in different bacterial contexts is influenced by changes in copy number, transcriptional and translational rates. iFFL based on broad host range promoters (PBHR promoter and PBHRsp1 promoter) has been demonstrated below to achieve controllable expression between E.coli and P.putida .

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 250
    Illegal XhoI site found at 3335
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 283
    Illegal PstI site found at 2538
    Illegal AgeI site found at 1277
  • 1000
    COMPATIBLE WITH RFC[1000]