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Team:BIOTEC Dresden/Project v2
From 2009.igem.org
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.
The project is split into three parts:
FLP Recombinase-based PoPS Measurement Device
Utilizing a new biobrick, the FLP reporter system can used to determine the transcription rate of gene expression. It can be applied to measure the persistence length of DNA by using different spacings between the FLP recombinase sites.
Instead of gene expression in cells, it is attempted to express this system in vitro, using lipid vesicles. First of all, a method to create those vesicles is introduced, then a gene expression kit is inserted.
Following the description given in ((add reference)), it is attempted to create nanoparticles using a silver-binding peptide. This is a promising approach for tagging proteins, but it turns out that the protocol is insufficient.
