Difference between revisions of "Part:BBa K2203002"
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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
 
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
  
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===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.
 +
M15 cells have a lacZ delta mutation which make then 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.
 +
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.
 +
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.
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<html>
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<figure>
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<img src="https://static.igem.org/mediawiki/parts/8/88/171002_CHARACTERIZATION_T7_LACZA_all.png" height="480" width="640">
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<figcaption> <center>Repeats of T7LacZalpha in T7M15 lysate </center></figcaption>
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</figure>
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</html>
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===Incubation reactions showing alpha complementation in T7-M15 cell lysate===
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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.
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Below the results after 1 hour of incubation:
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<html>
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<figure>
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<img src=https://static.igem.org/mediawiki/2017/e/e7/T--EPFL--page_1.png>
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<figcaption>1h incubation of T7 LacZalpha in T7 M15 lysate at 37°C</figcaption>
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</figure>
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</html>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Revision as of 11:20, 29 October 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 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 make then 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. 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. 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.

Repeats of T7LacZalpha in T7M15 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. Below the results after 1 hour of incubation:

1h incubation of T7 LacZalpha in T7 M15 lysate 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]