Improved Parts

Here's a list of parts we have improved in the scope of our project.

BBa_I732006 - LacZalpha

We improved the functionality of LacZalpha by modifying it in order to express LacZalpha in cell-free expression systems. On top of that we showed that Alpha Complementation could be achieved in our own E.Coli lysates. Alpha Complementation is a signaling pathway that makes use of a mutation found in certain E.Coli strains. The mutated strains contain a partial version of the LacZ gene. By adding the missing Alpha part as DNA template to a lysate reaction, a functional beta-galactosidase enzyme will be produced. Since the Alpha part has a size of 7.4% of the total LacZ gene, less resources are needed to synthesize the whole enzyme and transcription is sped up significantly. This translates into a faster and stronger colour change in our detection scheme and is thus a highly useful functionality.

To use this part in a cell-free environment, it needed a T7 promoter, since no expression is observed if a promoter is missing. We submitted our new composite BioBrick BBa_K2203002 and it can be found here.

At the same time we added another functionality to LacZalpha : It is the reporter gene in all of the toehold switches we used for our project. Thus we have coupled the mechanism of a toehold switch to alpha complementation and shown that it works as expected in our E.Coli lysates.

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

HCV Toehold A LacZalpha in T7 M15 lysate

Expression of LacZalpha was measured on a plate reader. Toehold and trigger were added to a usual lysate reaction as described here. Reactants were kept at 37°C for five hours.

Expression of reporter gene LacZalpha within HCV Toehold A

The toehold is successfully triggered and LacZalpha translated.

BBa_J64997 - T7 promoter

We improved this part by composing new BioBricks that all contain the T7 promoter. This promoter is well suited for cell-free expression systems as the T7 RNA polymerase is highly specific to this region and will efficiently translate any template having this promoter in front.

We characterized this part in cell extracts from different E.Coli strains and have successfully demonstrated that all composite parts work as expected.

The following composite parts made by us contain a T7 promoter : BBa_K2203001, BBa_K2203002, BBa_K2203003, BBa_K2203004, BBa_K2203005, BBa_K2203006.