Difference between revisions of "Team:IIT-Madras/Description"

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<h2 style="font-size: 9mm;">ADaPtat1on</h2>
 
<h2 style="font-size: 9mm;">ADaPtat1on</h2>
  
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<span style="padding-right: 77%;"><strong>Ideation:</strong></span>
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<span style="padding-right: 77%;"><strong>Motivations:</strong></span>
  
  
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<li>The original idea was to study the degradation of lignin to produce vanillin which has higher economic value.
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<li>Lignins are important in the formation of cell walls, especially in wood and bark, because they lend rigidity and do not rot easily. Lignin is rich in aromatic compounds which are degraded only by few organisms. Lot of work has been done on degrading aromatic compounds using Pseudomonas as chassis but even it has its own limitations. So, there is a pressing need to use other chassis that can complement the work. Acinetobacter baylyi ADP1 fits in exactly as due to its ability to degrade aromatic compounds and lignin-derived monomers.</li>
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We conducted a literature survey regarding the degradation of the aromatic rings of lignin and organisms capable of degrading such aromatics.</li>
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<li>For routine model organisms like E. coli and L. lactis., they are not naturally competent and so we need to artificially competent. We found many ‘Naturally Competent’ microorganisms. However, most of them are pathogenic and known to cause harm to humans. Hence, you cannot work with them in Biosafety level 1 lab. For example Streptococcus pneumoniae, Neisseria gonorrhoeae, Bacillus subtilis and Haemophilus influenzae.
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<li>For popular organisms like <em>E. coli</em> and <em>L. lactis.</em> competent cells are required for transformation. From a brief literature survey we found several ‘naturally competent’ microorganisms. However, most of them were pathogenic and are known to harm humans. Hence, we cannot work with them in a biosafety level 1 laboratory.
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<li>
 
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<span style="padding-right: 77%;"><strong>Solution:</strong></span>
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<span style="padding-right: 77%;"><strong>Background:</strong></span>
  
 
<p style="font-size:5.5mm; font-family: 'title', sans-serif;" class="p12 p16" ALIGN=LEFT >
 
<p style="font-size:5.5mm; font-family: 'title', sans-serif;" class="p12 p16" ALIGN=LEFT >
  
  
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An organism that best fits both these requirements is <em>Acinetobacter baylyi</em>. The ADP1 strain of this organism is naturally competent, non-pathogenic, and has the property of degrading aromatics. This strain can be purchased from DSMZ.
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We found from the literature that Acinetobacter baylyi ADP1 to be a good chassis for our project because of its ability to degrade aromatic compounds and its naturally competent. It is also non-pathogenic and belongs to risk group 1[1]. Also, recent work has been done to produce wax ester in Acinetobacter baylyi ADP1 [2]. Strains of Acinetobacter baylyi have been constructed that can utilize Gluconate and Glucose better than the existing strain [3].
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However, a big shortcoming of using this organism is that it does not many tools for gene manipulation. For example, usually, T5 and T7 are the only two standard promoters used in engineering this organism.
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<span style="padding-right: 75%;"><strong>Motivation:</strong></span>
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<span style="padding-right: 75%;"><strong>Project:</strong></span>
  
 
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A primary requirement for experimentation in synthetic biology is a fluorescent reporter protein. Hence we planned to codon-optimize two reporter proteins, GFP and mCherry, for <em>A. baylyi</em>. </p>
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Our objective is to create a T5 based synthetic promoter library for <em>Acinetobacter baylyi</em> ADP1. For this purpose, we also needed fluorescent reporter protein as we intended to measure the strength of promoter using fluorometry experiment. We approached GenScript for codon optimized GFP for <em>Acinetobacter baylyi</em> ADP1, however, they did not have the codon usage table data for <em>Acinetobacter baylyi</em>. 
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</em>. </p>
  
 
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When we approached companies for codon-optimized reporter proteins they did not have a codon bias table available for <em>A. baylyi</em>.Thus we made a freely available online tool called CUTE (Codon Usage Table Enumerator) that can generate a codon usage table by taking the protein coding annotation into consideration. This tool can be used for all organisms 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>  
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We made a free use online tool called CUTE(codon usage table easy) that can generate Codon usage table by taking into consideration the protein-coding annotation. This tool can be used for any other organism whose coding regions are annotated. Cute can be found on the <a href="https://cute.chassidex.org" target="_blank">cute.chassidex.org</a><br>  
  
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</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>
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We generated the codon usage table as the protein annotation of <em>Acinetobacter baylyi</em> ADP1 is available on the NCBI website. Using this table, we codon optimized GFP (which was codon optimized previously for E. coli). <br>
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</p>
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Next, we generated the T5 promoter-based library for Acinetobacter baylyi ADP1. Since these promoters are T5 based, they might potentially also work in other gram-negative organisms like E. coli strains, Cornybacterium etc.<br>
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Following this we conceptualised our next tool - a synthetic promoter library for <em>Acinetobacter baylyi</em> ADP1. We designed a T5 promoter-based library which can exhibit varying expression strengths. Our aim was to make this library as generic as possible so as it maximise its use in synthetic biology.
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Reference:<ol style="font-size:5.5mm; font-family: 'title', sans-serif;" class="p12 p16" ALIGN=LEFT>
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<li>https://www.dsmz.de/catalogues/details/culture/DSM-24193.html?tx_dsmzresources_pi5%5BreturnPid%5D=304</li>
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<li>Suvi Santala, Elena Efimova, Perttu Koskinen, Matti Tapani Karp, and Ville Santala ACS Synthetic Biology 2014 3 (3), 145-151 DOI: 10.1021/sb4000788</li>
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<li>Kannisto, Matti et al. “Metabolic Engineering of <em>Acinetobacter Baylyi</em> ADP1 for Improved Growth on Gluconate and Glucose.” Ed. S.-J. Liu. Applied and Environmental Microbiology 80.22 (2014): 7021–7027. PMC. Web. 17 Oct. 2018</li>
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Revision as of 23:10, 17 October 2018

iGEM Collaborations Page

☰

Team: IIT-Madras/ADaPtat1on

Description

ADaPtat1on

Motivations:

  1. Lignins are important in the formation of cell walls, especially in wood and bark, because they lend rigidity and do not rot easily. Lignin is rich in aromatic compounds which are degraded only by few organisms. Lot of work has been done on degrading aromatic compounds using Pseudomonas as chassis but even it has its own limitations. So, there is a pressing need to use other chassis that can complement the work. Acinetobacter baylyi ADP1 fits in exactly as due to its ability to degrade aromatic compounds and lignin-derived monomers.
  2. For routine model organisms like E. coli and L. lactis., they are not naturally competent and so we need to artificially competent. We found many ‘Naturally Competent’ microorganisms. However, most of them are pathogenic and known to cause harm to humans. Hence, you cannot work with them in Biosafety level 1 lab. For example Streptococcus pneumoniae, Neisseria gonorrhoeae, Bacillus subtilis and Haemophilus influenzae.

Background:

We found from the literature that Acinetobacter baylyi ADP1 to be a good chassis for our project because of its ability to degrade aromatic compounds and its naturally competent. It is also non-pathogenic and belongs to risk group 1[1]. Also, recent work has been done to produce wax ester in Acinetobacter baylyi ADP1 [2]. Strains of Acinetobacter baylyi have been constructed that can utilize Gluconate and Glucose better than the existing strain [3]. However, a big shortcoming of using this organism is that it does not many tools for gene manipulation. For example, usually, T5 and T7 are the only two standard promoters used in engineering this organism.

Project:

While perusing literature for Acinetobacter baylyi ADP1 we couldn't find substantial material, as information was sparse and not many tools were available to undertake synthetic biology experiments with this organism. This motivated us to build a toolbox for Acinetobacter baylyi that was generic and hence could effectively be used by research groups working with other organisms as well.

Project:

Our objective is to create a T5 based synthetic promoter library for Acinetobacter baylyi ADP1. For this purpose, we also needed fluorescent reporter protein as we intended to measure the strength of promoter using fluorometry experiment. We approached GenScript for codon optimized GFP for Acinetobacter baylyi ADP1, however, they did not have the codon usage table data for Acinetobacter baylyi. .

We made a free use online tool called CUTE(codon usage table easy) that can generate Codon usage table by taking into consideration the protein-coding annotation. This tool can be used for any other organism whose coding regions are annotated. Cute can be found on the cute.chassidex.org
We generated the codon usage table as the protein annotation of Acinetobacter baylyi ADP1 is available on the NCBI website. Using this table, we codon optimized GFP (which was codon optimized previously for E. coli).
Next, we generated the T5 promoter-based library for Acinetobacter baylyi ADP1. Since these promoters are T5 based, they might potentially also work in other gram-negative organisms like E. coli strains, Cornybacterium etc.
Reference:

  1. https://www.dsmz.de/catalogues/details/culture/DSM-24193.html?tx_dsmzresources_pi5%5BreturnPid%5D=304
  2. Suvi Santala, Elena Efimova, Perttu Koskinen, Matti Tapani Karp, and Ville Santala ACS Synthetic Biology 2014 3 (3), 145-151 DOI: 10.1021/sb4000788
  3. Kannisto, Matti et al. “Metabolic Engineering of Acinetobacter Baylyi ADP1 for Improved Growth on Gluconate and Glucose.” Ed. S.-J. Liu. Applied and Environmental Microbiology 80.22 (2014): 7021–7027. PMC. Web. 17 Oct. 2018

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