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Team:Warsaw/Project
From 2010.igem.org
Project description
Our project is divided in following parts:
- RBS Measurement
- Promoter Measurement
- MinC based kill switch
- BactoDHL - universal platform of protein and DNA delivery to mammalian cells based on invasive coli strain
RBS Measurement
In order to fine-tune expression of genes used in our project we have conducted measurement of various ribosome binding sites included in 2010 spring distribution. Our list of measured parts includes RBSs both from Community and Anderson's collections.
We have used standard measurement kit composed of promoter BBa_J23100 and GFP+terminator part BBa_I130401. All measurements were carried out using flow cytometer.
Results are here
We have used standard measurement kit composed of promoter BBa_J23100 and GFP+terminator part BBa_I130401. All measurements were carried out using flow cytometer.
Results are here
Promoter Measurement
Additionally we have defined new standard of absolute promoter strength measurement (RFC58).
Using it we have measured J23100 and pT7.
Results are here
Using it we have measured J23100 and pT7.
Results are here
Safety
To make our invasive E. coli strain safe we are planning to use division inhibitor protein MinC from E. coli BL21. Overexpression of MinC was shown to stop bacterial divisions without impairing the rest of cell functions, such as protein synthesis.
BactoDHL
BactoDHL is a universal platform of protein and DNA delivery to mammalian cells. It's based on E. coli strain that expresses invasion determinants: invasin from Yersinia pestis and listeriolysin (LLO) from Listeria monocytogenes. Invasin caused uptake of the bacterium into the mammalian cell by induction of clatrin-mediated endocytosis. Bacterial cells are lysed in the endosome and then LLO is relased. LLO is a pore-forming toxin which causes endosomal membrane disruption and release of the payload (either protein or DNA) into cytoplasm of the mammalian cell.
ColiStainer
We have planned to build a ColiStainer strain. Unfortunately there was not enough time to accomplish this task. Below is original information about ColiStainer concept.
ColiStainer is a BactoDHL strain expressing various fluorescence proteins (i.e. GFP) fused to various cell localization signals. When ColiStainer invades mammalian cells it releases payload of those proteins, which are transported to various cellular compartments (i.e. mitochondria, nucleus or Golgi apparatus). It causes the compartments to glow in various colors and produces cheaper and easier equivalent of immunohistochemical stain with florophore-conjugated antibodies.
ColiStainer is a BactoDHL strain expressing various fluorescence proteins (i.e. GFP) fused to various cell localization signals. When ColiStainer invades mammalian cells it releases payload of those proteins, which are transported to various cellular compartments (i.e. mitochondria, nucleus or Golgi apparatus). It causes the compartments to glow in various colors and produces cheaper and easier equivalent of immunohistochemical stain with florophore-conjugated antibodies.
