Contents
Synthetic biology with S. thermophilus
Escherichia coli is a commonly used chassis microorganism in synthetic biology because there is a wide range of information available regarding its characteristics and well-defined experimental protocols. Our ultimate aim was to improve the understanding of one of the lactic acid bacteria required in yogurt production: Streptococcus thermophilus. S. thermophilus could be incredibly useful in synthetic biology, specifically in creating yogurt with increased production of different macromolecules – this is an application where E. coli would not be suitable.
Although there are other bacteria present in yogurt (most importantly, Lactobacillus delbrueckii subsp. bulgaricus), we chose to use S. thermophilus because it is a naturally competent organism: certain strains of S. thermophilus are highly transformable – for example, the strain we used (LMD-9) has been shown to yield up to 106 transformants per ml of culture [1]. Competence can also be induced through adding a 24-amino-acid hydrophobic peptide (ComS) in strains that are not as easily transformed [2] This takes advantage of the ComRS system, whereby ComS associates with the Rgg-like regulator ComR in order to induce the transcription of comX. ComX encodes the sigma factor σX, which upregulates genes which are required for DNA transformation [2]. A protocol for transformation using ComS can be found on our protocols page.
The chassis subsection of our project has been split into three parts:
Parts submitted to the registry
Shuttle Vector
We have submitted a BioBrick compatible version of the shuttle vector to the Parts registry for future teams working with S. thermophilus (BBa_K2151666)
Native S. thermophilus promoters
We used three native S. thermophilus promoters during the course of the project. We made these BioBrick compatible and submitted them to the Parts registry for other teams to use in S.thermophilus based projects.
- BBa_K2151000: phlbA
- BBa_K2151001: p25
- BBa_K2151002 : p32
GFP-based BioBrick constructs
We ligated GFP with both strong and medium ribosome binding sites to the three native S. thermophilus promoters we used. These are useful reporter constructs for working with this organism.
- BBa_K2151006: phlbA ligated to I13500 (GFP with strong RBS)
- BBa_K2151007: p25 ligated to I13500 (GFP with strong RBS)
- BBa_K2151008 : p32 ligated to I13500 (GFP with strong RBS)
- BBa_K2151003: phlbA ligated to E5501 (GFP with medium RBS)
- BBa_K2151004: p25 ligated to E5501 (GFP with medium RBS)
- BBa_K2151005 : p32 ligated to E5501 (GFP with medium RBS)
amilCP
We ligated Uppsala's BioBrick BBa_K592025 to the three native S.thermophilus promoters we worked with throughout the project and submitted three new BioBricks. These could be utilised by teams working with S. thermophilus in order to expand upon the results we gathered.
- BBa_K2151009: phlbA ligated to BBa_K592025
- BBa_K2151010: p25 ligated to BBa_K592025
- BBa_K2151011 : p32 ligated to BBa_K592025
References
- ↑ Gardan, R., Besset, C., Guillot, A., Gitton, C. and Monnet, V. (2009). The Oligopeptide Transport System Is Essential for the Development of Natural Competence in Streptococcus thermophilus Strain LMD-9. Journal of Bacteriology, 191(14), pp.4647-4655.
- ↑ 2.0 2.1 Fontaine, L., Boutry, C., de Frahan, M., Delplace, B., Fremaux, C., Horvath, P., Boyaval, P. and Hols, P. (2009). A Novel Pheromone Quorum-Sensing System Controls the Development of Natural Competence in Streptococcus thermophilus and Streptococcus salivarius. Journal of Bacteriology, 192(5), pp.1444-1454.
