Difference between revisions of "Part:BBa K2970008"

 
 
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<partinfo>BBa_K2970008 short</partinfo>
 
<partinfo>BBa_K2970008 short</partinfo>
  
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It contains both triggers and the gate to test all three. (<partinfo>BBa_K2970000</partinfo>, <partinfo>BBa_K2970001</partinfo>, <partinfo>BBa_K2970002</partinfo>). The function of the gate is the regulation of the translation, which is activated by the triggers.
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To measure the functionality of our gate (<partinfo>BBa_K2970002</partinfo>, Figure 1B) and trigger (<partinfo>BBa_K2970000</partinfo> and <partinfo>BBa_K2970001</partinfo>) we designed a test plasmid which contains both trigger and the gate, which is locking GFP, each embedded into a promoter and a terminator. The promoters of the trigger can be induced by either IPTG or arabinose to test different combinations of trigger concentrations. In contrast, the gate is translated constitutively.
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The two triggers form a complex due to complementary areas. The sequence that is formed is complementary to the gate that in normal conformation forms a hairpin which covers the RBS and the start codon. If the trigger complex binds to the gate, this hairpin opens and the RBS is released and translation can take place. The affinity of the trigger complex is higher than the affinity of the gate within. The gate can only be opened if both triggers are present, otherwise the complementary region is too short. If the gate is opened, GFP is expressed whose signal can be measured.  
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The function of the gate is the regulation of the translation of a gene of interest, in this case GFP for testing, by formation of a hairpin by the mRNA that hides the ribosome binding site and the start codon. The mRNAs of the two triggers can form a complex due to complementarity (Figure 1A), which can open the gates hairpin and releases the ribosome binding site, thus translation can take place (Figure 2). The affinity of the trigger complex is higher than the affinity of the gate within. The gate can only be opened if both triggers are present, otherwise the complementary region is too short. If the gate is opened, GFP is expressed whose signal can be measured.
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<figure>
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<img width="100%" src="https://2019.igem.org/wiki/images/b/bc/T--Hamburg--PartsFigure11.jpeg">
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<figcaption>
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<b>Figure 1: </b>A) Formation of trigger complex after translation. B) mRNA of gate sequence forms secondary structures that hide the ribosome binding site and the start codon.
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</figcaption>
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</figure>
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</html>
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<html>
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<figure>
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<img width="90%" src="https://2019.igem.org/wiki/images/4/49/T--Hamburg--Part_Figure6.jpg">
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<figcaption>
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<b>Figure 2: </b>Opening of the gate due to annealing of trigger complex to gate.
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</figcaption>
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</figure>
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</html>
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===Results===
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Due to a high amount of secondary structures it was difficult to assemble this part. Therefore we could not provide characterization data.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 02:11, 22 October 2019


Gate/Trigger test composition

To measure the functionality of our gate (BBa_K2970002, Figure 1B) and trigger (BBa_K2970000 and BBa_K2970001) we designed a test plasmid which contains both trigger and the gate, which is locking GFP, each embedded into a promoter and a terminator. The promoters of the trigger can be induced by either IPTG or arabinose to test different combinations of trigger concentrations. In contrast, the gate is translated constitutively.

The function of the gate is the regulation of the translation of a gene of interest, in this case GFP for testing, by formation of a hairpin by the mRNA that hides the ribosome binding site and the start codon. The mRNAs of the two triggers can form a complex due to complementarity (Figure 1A), which can open the gates hairpin and releases the ribosome binding site, thus translation can take place (Figure 2). The affinity of the trigger complex is higher than the affinity of the gate within. The gate can only be opened if both triggers are present, otherwise the complementary region is too short. If the gate is opened, GFP is expressed whose signal can be measured.

Figure 1: A) Formation of trigger complex after translation. B) mRNA of gate sequence forms secondary structures that hide the ribosome binding site and the start codon.
Figure 2: Opening of the gate due to annealing of trigger complex to gate.

Results

Due to a high amount of secondary structures it was difficult to assemble this part. Therefore we could not provide characterization data.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 103
    Illegal NheI site found at 284
    Illegal NheI site found at 422
    Illegal NheI site found at 445
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 224
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1219