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 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 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

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]