Part:BBa_K2203002

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 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 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]