Difference between revisions of "Part:BBa K2203002"
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===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 | + | 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 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. | 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. | 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 | + | In fact beta-galactosidase is a tetramer, it needs four 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. | 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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Revision as of 17:11, 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 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.
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 four 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.
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:
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]