Difference between revisions of "Part:BBa I732006"
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<partinfo>BBa_I732006 SequenceAndFeatures</partinfo> | <partinfo>BBa_I732006 SequenceAndFeatures</partinfo> | ||
| − | ===Characterization | + | ===Characterization of 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 level of absorbance to use as a downstream reporter 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. | ||
<html> | <html> | ||
<figure> | <figure> | ||
Revision as of 10:54, 29 October 2017
lacZ alpha fragment
In strains with lacZ-omega (lacZ N-terminal deletion mutant) like DH5alpha, DH10B and Top10, lacZ-alpha restores the beta-galactosidase activity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization of 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 level of absorbance to use as a downstream reporter 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.
Improvements
This part was improved with a T7 promoter and terminator : BBa_K2203002 . This part is also used as a reporter in the following composite parts which are toehold switches : BBa_K2203003 , BBa_K2203004 , BBa_K2203005 , BBa_K2203006 .