Difference between revisions of "Team:Glasgow/Chassis"

 
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Team Members on this side of the project: Katy and Mat
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==Synthetic biology with S. thermophilus==
  
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We aim to characterise Streptococcus thermophilus in order to increase understanding and ability to work with this potentially useful organism. By transforming S. thermophilus with a range of reporters under the promotion of different Anderson promoters and characterising these parts, we hope to provide future teams with a good starting ground to working with this yogurt bacteria.  
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<i>Escherichia coli</i> 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.
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We will also write protocols for working with S. thermophilus.
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Our ultimate aim was to improve the understanding of one of the lactic acid bacteria required in yogurt production: <i>Streptococcus thermophilus</i>. <i>S. thermophilus</i> could be incredibly useful in synthetic biology, specifically in creating yogurt with increased production of different macromolecules – this is an application where <i>E. coli</i> would not be suitable.  
  
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Although there are other bacteria present in yogurt (most importantly, <i>Lactobacillus delbrueckii subsp. bulgaricus</i>), we chose to use <i>S. thermophilus</i> because it is a naturally competent organism: certain strains of <i>S. thermophilus</i> are highly transformable – for example, the strain we used (LMD-9) has been shown to yield up to 10<sup>6</sup> transformants per ml of culture <ref>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.
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</ref>. Competence can also be induced through adding a 24-amino-acid hydrophobic peptide (ComS) in strains that are not as easily transformed <ref name="Fontaine">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.</ref> This takes advantage of the ComRS system, whereby ComS associates with the Rgg-like regulator ComR in order to induce the transcription of <i>comX</i>. <i>ComX</i> encodes the sigma factor σX, which upregulates genes which are required for DNA transformation <ref name="Fontaine"/>. A protocol for transformation using ComS can be found on our [https://2016.igem.org/Team:Glasgow/Protocols protocols page].
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The chassis subsection of our project has been split into three parts:
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<ul>
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<li>[https://2016.igem.org/Team:Glasgow/ShuttleVector Shuttle Vector]</li>
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<li>[https://2016.igem.org/Team:Glasgow/Description Characterising amilCP]</li>
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<li>[https://2016.igem.org/Team:Glasgow/Measurement Quantification of promoter strength]</li></ul>
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==Parts submitted to the registry==
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===Shuttle Vector===
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We have submitted a BioBrick compatible version of the shuttle vector to the Parts registry for future teams working with <i>S. thermophilus</i> ([http://parts.igem.org/Part:BBa_K2151666 BBa_K2151666])
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===Native <i>S. thermophilus</i> promoters===
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We used three native <i>S. thermophilus</i> 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 <i>S.thermophilus</i> based projects.
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<ul>
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<li><b>[http://parts.igem.org/Part:BBa_K2151000 BBa_K2151000]</b>: phlbA
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<li><b>[http://parts.igem.org/Part:BBa_K2151001 BBa_K2151001]</b>: p25
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<li><b>[http://parts.igem.org/Part:BBa_K2151002 BBa_K2151002] </b>: p32
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</ul>
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===GFP-based BioBrick constructs===
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We ligated GFP with both strong and medium ribosome binding sites to the three native <i>S. thermophilus</i> promoters we used. These are useful reporter constructs for working with this organism.
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<ul>
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<li><b>[http://parts.igem.org/Part:BBa_K2151006 BBa_K2151006]</b>: phlbA ligated to I13500 (GFP with strong RBS)
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<li><b>[http://parts.igem.org/Part:BBa_K2151007 BBa_K2151007]</b>: p25 ligated to I13500 (GFP with strong RBS)
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<li><b>[http://parts.igem.org/Part:BBa_K2151008 BBa_K2151008] </b>: p32 ligated to I13500 (GFP with strong RBS)
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<li><b>[http://parts.igem.org/Part:BBa_K2151003 BBa_K2151003]</b>: phlbA ligated to E5501 (GFP with medium RBS)
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<li><b>[http://parts.igem.org/Part:BBa_K2151004 BBa_K2151004]</b>: p25 ligated to E5501 (GFP with medium RBS)
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<li><b>[http://parts.igem.org/Part:BBa_K2151005 BBa_K2151005] </b>: p32 ligated to E5501 (GFP with medium RBS)
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</ul>
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===amilCP===
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We ligated Uppsala's BioBrick [http://parts.igem.org/Part:BBa_K592025 BBa_K592025] to the three native <i>S.thermophilus</i> promoters we worked with throughout the project and submitted three new BioBricks. These could be utilised by teams working with <i>S. thermophilus</i> in order to expand upon the results we gathered.
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<ul>
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<li><b>[http://parts.igem.org/Part:BBa_K2151009 BBa_K2151009]</b>: phlbA ligated to [http://parts.igem.org/Part:BBa_K592025 BBa_K592025]
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<li><b>[http://parts.igem.org/Part:BBa_K2151010 BBa_K2151010]</b>: p25 ligated to [http://parts.igem.org/Part:BBa_K592025 BBa_K592025]
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<li><b>[http://parts.igem.org/Part:BBa_K2151011 BBa_K2151011] </b>: p32 ligated to [http://parts.igem.org/Part:BBa_K592025 BBa_K592025]
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</ul>
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==References==
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<small><references/></small>
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Latest revision as of 22:51, 19 October 2016

Glasgow iGEM 2016
Chassis

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 ([http://parts.igem.org/Part:BBa_K2151666 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.

  • [http://parts.igem.org/Part:BBa_K2151000 BBa_K2151000]: phlbA
  • [http://parts.igem.org/Part:BBa_K2151001 BBa_K2151001]: p25
  • [http://parts.igem.org/Part:BBa_K2151002 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.

  • [http://parts.igem.org/Part:BBa_K2151006 BBa_K2151006]: phlbA ligated to I13500 (GFP with strong RBS)
  • [http://parts.igem.org/Part:BBa_K2151007 BBa_K2151007]: p25 ligated to I13500 (GFP with strong RBS)
  • [http://parts.igem.org/Part:BBa_K2151008 BBa_K2151008] : p32 ligated to I13500 (GFP with strong RBS)
  • [http://parts.igem.org/Part:BBa_K2151003 BBa_K2151003]: phlbA ligated to E5501 (GFP with medium RBS)
  • [http://parts.igem.org/Part:BBa_K2151004 BBa_K2151004]: p25 ligated to E5501 (GFP with medium RBS)
  • [http://parts.igem.org/Part:BBa_K2151005 BBa_K2151005] : p32 ligated to E5501 (GFP with medium RBS)


amilCP

We ligated Uppsala's BioBrick [http://parts.igem.org/Part:BBa_K592025 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.

  • [http://parts.igem.org/Part:BBa_K2151009 BBa_K2151009]: phlbA ligated to [http://parts.igem.org/Part:BBa_K592025 BBa_K592025]
  • [http://parts.igem.org/Part:BBa_K2151010 BBa_K2151010]: p25 ligated to [http://parts.igem.org/Part:BBa_K592025 BBa_K592025]
  • [http://parts.igem.org/Part:BBa_K2151011 BBa_K2151011] : p32 ligated to [http://parts.igem.org/Part:BBa_K592025 BBa_K592025]


References

  1. ↑ 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. ↑ 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.