Difference between revisions of "Team:IIT-Madras/Description"
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<ol type="I" style="font-size: 5.5mm; text-align: justify; " ALIGN=LEFT> | <ol type="I" style="font-size: 5.5mm; text-align: justify; " ALIGN=LEFT> | ||
| − | <li> | + | <li>The initial idea was lignin degradation to produce higher-valued vanillin. |
| − | We looked up the literature | + | We looked up the literature regarding the degradation of the aromatic rings of lignin. We needed an organism that could degrade aromatics.</li> |
| − | <li>For popular | + | <li>For popular organisms like <em>E.coli</em> and <em>L. lactis.</em>, we need to make competent cells for transformation. We looked up the surrounding literature and found several ‘naturally competent’ microorganisms. However, most of them are pathogenic and are known to cause harm to humans. Hence, they cannot be worked with in a biosafety level 1 lab. For example, <em>Streptococcus pneumoniae</em> (Griffith's "pneumococcus"), <em>Neisseria gonorrhoeae</em>, <em>Bacillus subtilis</em> and <em>Haemophilus influenzae</em>.</li> |
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| − | The organism that best fits | + | The organism that best fits both these requirements is <em>Acinetobacter baylyi</em> ADP1 strain. This organism is naturally competent, non-pathogenic, and has the amazing property of degrading aromatics. The strain can be purchased from DSMZ. |
</p> | </p> | ||
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| − | We looked up the literature for <em>Acinetobacter baylyi</em> ADP1 but could not find substantial | + | We looked up the literature for <em>Acinetobacter baylyi</em> ADP1 but could not find substantial material. The literature was too sparse and not many tools were available to undertake synthetic biology experiments with this organism. This was the motivation to generate a toolbox for <em>Acinetobacter baylyi</em> and to try and make the tools as generic as possible such that any teams or research groups wishing to work with other organisms could still use them. |
</p> | </p> | ||
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| − | The first thing that is required for any synthetic biology experiments | + | The first thing that is required for any synthetic biology experiments is a fluorescent reporter protein. We wished to codon-optimize GFP and mCherry for <em>A. baylyi</em>. However, when we asked the companies to codon-optimize the fluorescent proteins, the companies did not have the codon bias table for <em>A. baylyi</em>.<br><br> |
| − | <strong>Solution 1:</strong> We made a free use online tool called CUTE ( | + | <strong>Solution 1:</strong> We made a free-to-use online tool called CUTE (Codon Usage Table Enumerator) that can generate a codon usage table by taking into consideration the protein coding annotation. This tool can be used for any other organism whose genome has been sequenced and whose protein coding regions are annotated. CUTE can be found on the <a href="https://cute.chassidex.org" target="_blank">CUTE ChassiDex website.</a><br>We generated the codon usage table from the protein annotation of <em>A. baylyi</em> available on the <a href="https://ncbi.nlm.nih.gov" target="_blank">NCBI</a>website. Using this codon table, we codon optimized reporter proteins and the characterization can be found here. <br><br> |
| − | <strong>Solution 2:</strong> Next we | + | <strong>Solution 2:</strong> Next we moved on to building a synthetic promoter library for <em>Acinetobacter baylyi</em> ADP1, but we wanted to make the promoter library as generic as possible. So, we made a T5 promoter-based library which exhibits varying strengths. This library can be extremely useful for metabolic engineering and synthetic biology experiments.<br> |
Revision as of 14:29, 17 October 2018
Description
ADaPtat1on
Motivations:
- The initial idea was lignin degradation to produce higher-valued vanillin. We looked up the literature regarding the degradation of the aromatic rings of lignin. We needed an organism that could degrade aromatics.
- For popular organisms like E.coli and L. lactis., we need to make competent cells for transformation. We looked up the surrounding literature and found several ‘naturally competent’ microorganisms. However, most of them are pathogenic and are known to cause harm to humans. Hence, they cannot be worked with in a biosafety level 1 lab. For example, Streptococcus pneumoniae (Griffith's "pneumococcus"), Neisseria gonorrhoeae, Bacillus subtilis and Haemophilus influenzae.
The organism that best fits both these requirements is Acinetobacter baylyi ADP1 strain. This organism is naturally competent, non-pathogenic, and has the amazing property of degrading aromatics. The strain can be purchased from DSMZ.
Motivations:We looked up the literature for Acinetobacter baylyi ADP1 but could not find substantial material. The literature was too sparse and not many tools were available to undertake synthetic biology experiments with this organism. This was the motivation to generate a toolbox for Acinetobacter baylyi and to try and make the tools as generic as possible such that any teams or research groups wishing to work with other organisms could still use them.
Project:
The first thing that is required for any synthetic biology experiments is a fluorescent reporter protein. We wished to codon-optimize GFP and mCherry for A. baylyi. However, when we asked the companies to codon-optimize the fluorescent proteins, the companies did not have the codon bias table for A. baylyi.
Solution 1: We made a free-to-use online tool called CUTE (Codon Usage Table Enumerator) that can generate a codon usage table by taking into consideration the protein coding annotation. This tool can be used for any other organism whose genome has been sequenced and whose protein coding regions are annotated. CUTE can be found on the CUTE ChassiDex website.
We generated the codon usage table from the protein annotation of A. baylyi available on the NCBIwebsite. Using this codon table, we codon optimized reporter proteins and the characterization can be found here.
Solution 2: Next we moved on to building a synthetic promoter library for Acinetobacter baylyi ADP1, but we wanted to make the promoter library as generic as possible. So, we made a T5 promoter-based library which exhibits varying strengths. This library can be extremely useful for metabolic engineering and synthetic biology experiments.