Difference between revisions of "Part:BBa K2203004"

 
 
(5 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2203004 short</partinfo>
 
<partinfo>BBa_K2203004 short</partinfo>
  
−
Sequence of toehold 27B detecting a part of the Zika virus. The coding sequence for lacZalpha has been added after the toehold as a reporter protein. This part can now be used in combination with cell-free extracts to detect the presence of Zika virus in a sample.
+
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===
 +
<html>
 +
<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>
 +
<center>
 +
 
 +
<figure>
 +
<img src="https://static.igem.org/mediawiki/parts/9/9b/27B.png" height="480" width="640">
 +
<figcaption> <center>27B_Zika Toehold in T7M15 lysate </center></figcaption>
 +
</figure>
 +
</center>
 +
 
 +
<p> The toehold is successfully triggered and LacZalpha translated.</p>
 +
</html>
 +
 
 +
 
 +
===Incubation reaction of 27B_Zika Toehold in T7M15 lysate in the presence of a single stranded DNA trigger===
 +
 
 +
<html>
 +
<center>
 +
  <figure>
 +
              <img src=https://static.igem.org/mediawiki/2017/c/c9/T--EPFL--page_2.png>
 +
              <figcaption>Left : Toehold lacZ alpha triggered by the addition of ssDNA complementary sequence in T7 M15 lysates <br> Right: Toehold LacZ alpha with no trigger added: the alpha part cannot be translated </figcaption>             
 +
              </figure>
 +
</center>
 +
</html>
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

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]