Team:BIOTEC Dresden/Project v2

Project Description

Temporal and spatial control of protein synthesis by in vitro recombination inside picoliter reactors

Manufacturing functionalized proteins in vitro poses a challenge, as it requires coordinated molecular assemblies and multi-step reactions. In this project we aim to control, over time and space, the production of proteins tagged with a silver-binding peptide for in situ silver nanoparticle nucleation inside microdroplets generated by microfluidic devices.

Combining a transcription-translation system with protein coding genes and a recombination logic inside microdroplets provides spatial control. Moreover, in the microfluidic chamber we can pinpoint the beginning of synthesis, and easily track and isolate the droplets. Site-specific recombination generates a molecular timer for temporal control of protein synthesis.

Unlike transcriptional regulation, this method gives true all-or-none induction due to covalent modification of DNA by Flp recombinase. Determining the transfer curve of inter-FRT site distance versus average recombination time allows the onset of gene expression to be predicted. We then apply this Flp reporter system as a powerful PoPS measurement device.

Realization

The project is split into three parts:

FLP Recombinase-based genetic timer

A genetic timer was designed by characterizing the correlation between recombination efficiency of Flp recombinase and the distance between the FRT sites it recognizes. The transfer curve thus achieved would allow us to predict the onset of expression of our reporter. A useful application would be a PoPs measurement device.

Vesicle formation in a microfluidic chamber

Instead of gene expression in cells, it is attempted to place a in-vitro transcription-translation systemin vesicles. First of all, a method to create those vesicles is introduced, gene expression experiments inside the vesicles are still underway.

Metallization of a protein with silver

In this part of the project we wanted to metallize a protein with silver. For this we used a peptide sequence known to nucleate the formation of silver nanoparticles in silver-resistant bacteria.