Difference between revisions of "Part:BBa K2203004"
 
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<partinfo>BBa_K2203004 short</partinfo>
 
<partinfo>BBa_K2203004 short</partinfo>
  
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This composite part consists of a T7 promoter (BBa_J64997) in front of the toehold, a lacZalpha (BBa_I732006) downstream of it and a T7 terminator (BBa_K731721) at the end of it.
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This composite part consists of a T7 promoter (BBa_J64997) in front of the toehold, lacZalpha (BBa_I732006) downstream and a T7 terminator (BBa_K731721).
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Sequence of toehold 27B detecting a part of the Zika virus. This part can now be used in combination with cell-free extracts to detect the presence of Zika virus in a sample.
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The sequence of toehold 27B is detecting a part of the Zika virus. This part can now be used in combination with cell-free extracts to detect the presence of Zika virus in a sample.
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The reporter system in our toeholds(K2203004, K2203005 for zika detection and K2203002, K2203006 for hepatitis C detection works as follows:  
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The reporter system in our toeholds(K2203004, K2203005 for zika detection and K2203003, K2203006 for hepatitis C detection) works as follows:  
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- M15 cells have a lacZ delta mutation which makes them encode a form of beta-galactosidase lacking residues 11-41.  
 
- M15 cells have a lacZ delta mutation which makes them encode a form of beta-galactosidase lacking residues 11-41.  
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- Beta-galactosidase produced without those residues is missing a small part and is thus not functional.  
 
- Beta-galactosidase produced without those residues is missing a small part and is thus not functional.  
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- Upon the annealing of a trigger zika sequence to the toehold switch, the toehold unfolds and the lacZalpha coding sequence is translated.  
 
- Upon the annealing of a trigger zika sequence to the toehold switch, the toehold unfolds and the lacZalpha coding sequence is translated.  
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- The production of the lacZaplpha part and the indigenous presence of the lacZ omega part in M15 cells allows for alpha complementation and production of a functional beta-galactosidase that can be detected by the addition of a colorimetric substrate CPRG Chlorophenol red-β-D-galactopyranoside.
 
- The production of the lacZaplpha part and the indigenous presence of the lacZ omega part in M15 cells allows for alpha complementation and production of a functional beta-galactosidase that can be detected by the addition of a colorimetric substrate CPRG Chlorophenol red-β-D-galactopyranoside.
  
 
===Characterization of the 27B_Zika Toehold in T7M15 lysate in the presence of a single stranded DNA trigger===
 
===Characterization of the 27B_Zika Toehold in T7M15 lysate in the presence of a single stranded DNA trigger===
 
<html>
 
<html>
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<p> Expression of LacZalpha was measured on a plate reader. Toehold and trigger were added to a usual lysate reaction as described <a href="http://2017.igem.org/Team:EPFL/Protocols#platereader"  target="_blank"><font color='blue'>here</font></a>. Reactants were kept at 37°C for five hours. </p>
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<center>
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  <figure>
 
  <figure>
 
<img src="https://static.igem.org/mediawiki/parts/9/9b/27B.png" height="480" width="640">
 
<img src="https://static.igem.org/mediawiki/parts/9/9b/27B.png" height="480" width="640">
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<figcaption> <center>Repeats of T7LacZalpha in T7M15 lysate </center></figcaption>
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<figcaption> <center>27B_Zika Toehold in T7M15 lysate </center></figcaption>
 
</figure>
 
</figure>
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</center>
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<p> The toehold is successfully triggered and LacZalpha translated.</p>
 
</html>
 
</html>
  

Latest revision as of 17:37, 29 October 2017


ZIKV_27B_lacZalpha

This composite part consists of a T7 promoter (BBa_J64997) in front of the toehold, lacZalpha (BBa_I732006) downstream and a T7 terminator (BBa_K731721). The sequence of toehold 27B is detecting a part of the Zika virus. This part can now be used in combination with cell-free extracts to detect the presence of Zika virus in a sample.

The reporter system in our toeholds(K2203004, K2203005 for zika detection and K2203003, K2203006 for hepatitis C detection) works as follows:

- M15 cells have a lacZ delta mutation which makes them encode a form of beta-galactosidase lacking residues 11-41.

- Beta-galactosidase produced without those residues is missing a small part and is thus not functional.

- Upon the annealing of a trigger zika sequence to the toehold switch, the toehold unfolds and the lacZalpha coding sequence is translated.

- The production of the lacZaplpha part and the indigenous presence of the lacZ omega part in M15 cells allows for alpha complementation and production of a functional beta-galactosidase that can be detected by the addition of a colorimetric substrate CPRG Chlorophenol red-β-D-galactopyranoside.

Characterization of the 27B_Zika Toehold in T7M15 lysate in the presence of a single stranded DNA trigger

Expression of LacZalpha was measured on a plate reader. Toehold and trigger were added to a usual lysate reaction as described here. Reactants were kept at 37°C for five hours.

27B_Zika Toehold in T7M15 lysate

The toehold is successfully triggered and LacZalpha translated.


Incubation reaction of 27B_Zika Toehold in T7M15 lysate in the presence of a single stranded DNA trigger

Left : Toehold lacZ alpha triggered by the addition of ssDNA complementary sequence in T7 M15 lysates
Right: Toehold LacZ alpha with no trigger added: the alpha part cannot be translated


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]