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Team:Uppsala/chassi
From 2013.igem.org
Chassi
Lactobacillus as the new probiotic chassi for iGEM
There are many challenges for anyone who wants to do synthetic biology in a new less common organism, like getting familiar with new protocols and techniques. But another major problem is that there is a lack of fundamental parts available like plasmids, reporter genes and promoters. A big part of our project has been to develop these basic parts and make them biobrick compatible. This is done in order to open up the Lactobacillus genus as the new probiotic chassi of choice for the iGEM community. We also intend to use it ourselves for doing metabolic engineering in Lactobacillus. For this we have worked to create two shuttle vectors , a library of constitutive promoters and a couple of reporter genes that should be functional in both E. coli and Lactobacillus. E. coli is substantially easier and faster to work with. Therefore the intention is to allow construction and some characterisation to first be done in E. coli, the construct can then later be transformed to Lactobacillus.
Promoters
Different constructs need different levels of expression. We have constructed and characterised a library of constitutive promoters of different strengths. These are based on a consensus sequence from promoters in Lactococcus lactis but have also been shown to work very well in E. coli and Lactobacillus.
Reporter genes
Many techniques and methods require fluorescent reporter genes to function. While some fluorescent proteins work poorly in Lactobacillus we have biobricked a codon-optimized version of mCherry that has been shown to work. This has been done according to assembly standard 25 to allow it to be used as a fusion protein.
Toxin-antitoxin
A major challenge in synthetic biology is that there is a selective pressure against metabolically taxing systems. In nature, many natural plasmids contain genes coding for a toxin and the associated antitoxin. These systems can significantly increase the segregational stability of plasmids and is an easy to use alternative to chromosomal integration for some applications.
Vectors
Perhaps the most important chassi parts we have constructed are shuttle vectors that work both in Lactobacillus and E. coli. Because Lactobacillus is significantly harder to work with and ligations are hard to transform, constructs should first be assembled and prepped in E. coli and then transferred to Lactobacillus with the shuttle vector.
Signal peptides
Lactobacillus is a great genus for the secretion of proteins and contains several secretory pathways. In order for a protein to be exported it needs to be tagged with a short amino acid sequence at the N-terminus called a signal peptide. We have synthesised a signal peptide that can be assembled with a protein of choice.
