Difference between revisions of "Part:BBa K2203002"
(→‎Incubation reactions showing alpha complementation in T7-M15 cell lysate)
 
(8 intermediate revisions by 2 users not shown)
Line 7: Line 7:
  
 
===Characterization of T7-lacZalpha in a cell-free chassis===
 
===Characterization of T7-lacZalpha in a cell-free chassis===
−
In order to improve the characterization of the lacZalpha fragment, we characterized it in a T7-M15 cell lysate to see whether we obtain high levels of absorbance to use beta-galactosidase and alpha complemetation as our downstream reporter scheme in further experiments.
+
In order to improve the characterization of the T7 LacZalpha fragment, we characterized it in a T7-M15 cell lysate to see whether we obtain high levels of absorbance to use beta-galactosidase and alpha complemetation as our downstream reporter scheme in further experiments.
−
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, resulting in a form of beta-galactosidase lacking residues 11-41. Beta-galactosidase produced without those residues is missing a small part as it does not produce the alpha subunit and is thus not functional. But if this mutated form of beta-galactosidase is brought together with the missing lacZ alpha part, the two will connect and form a functional beta-galactosidase part. The missing lacZalpha part is expressed in the lysate in this case.
  
−
Beta-galactosidase produced without those residues is missing a small part and is thus not functional.
+
In fact, beta-galactosidase is a tetramer, which means it is made up of four identical single units. Each unit needs one LacZalpha part to work.
−
But if this mutated form of beta-galactosidase is brought together with the missing lacZ alpha part (which we express in the lysate in this case), the two will connect and form a functional beta-galactosidase part.
+
The figure below shows the expression of a functional beta-galactosidase in T7-M15 cells  upon the assembly of the differents alpha and omega parts. For details on how the lysate and the energy solution were made and which components went into the final reaction volume of 10uL, check out our [http://2017.igem.org/Team:EPFL/Protocols protocols].  
−
In fact beta-galactosidase is a tetramer, it needs for units of LacZalpha and the rest to assemble in order to function.
+
−
The figure below shows the expression of a functional beta-galactosidase in T7-M15 cells  upon the assembly of the differents alpha and omega parts.
+
 
<html>
 
<html>
 
  <figure>
 
  <figure>
Line 22: Line 20:
  
 
===Incubation reactions showing alpha complementation in T7-M15 cell lysate===
 
===Incubation reactions showing alpha complementation in T7-M15 cell lysate===
−
We perfromed incubation reactions at 37C of the DNA template in lysate and a no DNA control adding a colorometric substrate to detect the presence of functional beta-galactosidase: CPRG Chlorophenol red-β-D-galactopyranoside.
+
We performed incubation reactions at 37°C: DNA template was added to lysate with a no DNA control. Both reaction contained a colorometric substrate to detect the presence of functional beta-galactosidase as it induces a color change when beta-galactosidase acts on it: CPRG Chlorophenol red-β-D-galactopyranoside.
−
Below the results after 1 hour of incubation:
+
Below are the results after 1 hour of incubation:
 
<html>
 
<html>
−
<center>
+
 
 +
<figure>
 +
<img style="width:30%" img align="center" img src="https://static.igem.org/mediawiki/parts/4/46/T--EPFL--page_alpha-turned.jpg" >
 +
<figcaption>Figure 1a: 1h incubation of T7 LacZalpha in T7 M15 lysate at 37°C</figcaption>
 +
</figure>
 +
 
 +
 
 +
 
 
<figure>
 
<figure>
−
<img src=https://static.igem.org/mediawiki/2017/e/e7/T--EPFL--page_1.png>
+
<img style="width:30%" img align="center" img src="https://static.igem.org/mediawiki/parts/3/3c/T--EPFL--page_alpha-control.jpg">
−
<figcaption>1h incubation of T7 LacZalpha in T7 M15 lysate at 37°C</figcaption>
+
<figcaption>Figure 1b: 1h incubation of no DNA control at 37°C</figcaption>
 
</figure>
 
</figure>
−
</center>
 
  
 
</html>
 
</html>

Latest revision as of 02:23, 2 November 2017


T7-lacZalpha

Improvement of the part BBa_I732006 by adding a T7 promoter (BBa_J64997) in front of the coding sequence and a T7 terminator (BBa_K731721) at the end of it to make it applicable for cell-free expression systems containing T7 polymerase


Characterization of T7-lacZalpha in a cell-free chassis

In order to improve the characterization of the T7 LacZalpha fragment, we characterized it in a T7-M15 cell lysate to see whether we obtain high levels of absorbance to use beta-galactosidase and alpha complemetation as our downstream reporter scheme in further experiments. M15 cells have a lacZ delta mutation, resulting in a form of beta-galactosidase lacking residues 11-41. Beta-galactosidase produced without those residues is missing a small part as it does not produce the alpha subunit and is thus not functional. But if this mutated form of beta-galactosidase is brought together with the missing lacZ alpha part, the two will connect and form a functional beta-galactosidase part. The missing lacZalpha part is expressed in the lysate in this case.

In fact, beta-galactosidase is a tetramer, which means it is made up of four identical single units. Each unit needs one LacZalpha part to work. The figure below shows the expression of a functional beta-galactosidase in T7-M15 cells upon the assembly of the differents alpha and omega parts. For details on how the lysate and the energy solution were made and which components went into the final reaction volume of 10uL, check out our [http://2017.igem.org/Team:EPFL/Protocols protocols].

Repeats of T7LacZalpha in T7M15 lysate

Incubation reactions showing alpha complementation in T7-M15 cell lysate

We performed incubation reactions at 37°C: DNA template was added to lysate with a no DNA control. Both reaction contained a colorometric substrate to detect the presence of functional beta-galactosidase as it induces a color change when beta-galactosidase acts on it: CPRG Chlorophenol red-β-D-galactopyranoside. Below are the results after 1 hour of incubation:

Figure 1a: 1h incubation of T7 LacZalpha in T7 M15 lysate at 37°C
Figure 1b: 1h incubation of no DNA control at 37°C
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]