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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<h4 align="left"><strong>Motivations:</strong></h4>
  
  
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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><i>Acinetobacter baylyi</i> ADP1 has catabolic pathways utilised in degrading aromatic compounds. These pathways can be re-engineered to produce biofuels from lignin monomers which are abundant in plant biomass. It can also be useful in producing bio-surfactants and lubricants such as Wax-Ester (Kannisto et al. 2016, Journal of Industrial Microbiology and Biotechnology). These pathways are not present in many other organisms. Some work has been done with regard to degrading aromatic compounds using <i>Pseudomonas</i> as the chassis but this has its limitations. <i>Acinetobacter baylyi</i> ADP1 complements the abilities and applications of <i>Pseudomonas</i>. </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>Routine model organisms like <i>E. coli</i> and <i>L. lactis</i> are not naturally competent. We have found many other ‘Naturally Competent’ microorganisms like <i>Streptococcus pneumoniae, Neisseria gonorrhoeae, Bacillus subtilis</i> and <i>Haemophilus influenzae</i>. However, most of them are pathogenic. Thus it is not possible work with them in a Biosafety level 1 laboratory.  
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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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<br><br>
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<span style="padding-right: 77%;"><strong>Solution:</strong></span>
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<h4 align="left">Background:</strong></h4>
  
 
<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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Literature studies indicate that Acinetobacter baylyi ADP1 is a good chassis for our project because of its ability to degrade aromatic compounds and its naturally competency. It is non-pathogenic and belongs to risk group 1<sup><a href="#1">[1]</a></sup>. Recent work has been done to produce wax ester in Acinetobacter baylyi ADP1 <sup><a href="#1">[2]</a></sup>. Strains of <em> Acinetobacter baylyi </em> have been engineered that can utilize Gluconate and Glucose more efficiently than existing strains. <sup><a href="#1">[3]</a></sup>.
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However, a major shortcoming of this organism is that not many tools are available for genetic engineering. For example, typically only T5 and T7 are the two standard promoters used in engineering this organism.
  
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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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<br><br>
  
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<h4 align="left">Project:</strong></span></h4>
  
  
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<span style="padding-right: 75%;"><strong>Motivation:</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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Our objective was to create a T5 based synthetic promoter library for <em>Acinetobacter baylyi</em> ADP1. A reporter protein was required to measure the strength of promoter using fluorometry. We had approached GenScript for codon optimized GFP for <em>Acinetobacter baylyi</em> ADP1. Reliable codon usage table data for <em>Acinetobacter baylyi</em> was not available. 
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</em>.
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Hence, we made a free-to-use online tool called CUTE (codon usage table easy) that can generate a codon usage table by taking into consideration the genomic protein-coding annotation. This tool can be used for any organism whose coding regions are annotated in the genome. Cute can be found at <a href="https://cute.chassidex.org" target="_blank">cute.chassidex.org</a><br> 
  
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While perusing literature for <em>Acinetobacter baylyi</em> 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 <em>Acinetobacter baylyi</em> that was generic and hence could effectively be used by research groups working with other organisms as well.
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Using this tool, we generated a codon usage table from the protein annotation data of <em>Acinetobacter baylyi</em> ADP1 which 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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Following this, we generated a T5 promoter-based library for <em>Acinetobacter baylyi</em> ADP1. Since these promoters are T5 based, they could potentially work in other gram-negative organisms like E. coli strains, Cornybacterium etc.<br><br><br>
  
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<span style="padding-right: 80%;"><strong>Project:</strong></span>
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<p id="1"></p>
  
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<p style="font-size:5.5mm; font-family: 'title', sans-serif;" class="p12 p16" ALIGN=LEFT >
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<h4 align="left"><strong>References:</strong></h4>
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<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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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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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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</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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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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Latest revision as of 15:09, 6 December 2018

iGEM Collaborations Page

☰

Team: IIT-Madras/ADaPtat1on

Description

ADaPtat1on

Motivations:

  1. Acinetobacter baylyi ADP1 has catabolic pathways utilised in degrading aromatic compounds. These pathways can be re-engineered to produce biofuels from lignin monomers which are abundant in plant biomass. It can also be useful in producing bio-surfactants and lubricants such as Wax-Ester (Kannisto et al. 2016, Journal of Industrial Microbiology and Biotechnology). These pathways are not present in many other organisms. Some work has been done with regard to degrading aromatic compounds using Pseudomonas as the chassis but this has its limitations. Acinetobacter baylyi ADP1 complements the abilities and applications of Pseudomonas.
  2. Routine model organisms like E. coli and L. lactis are not naturally competent. We have found many other ‘Naturally Competent’ microorganisms like Streptococcus pneumoniae, Neisseria gonorrhoeae, Bacillus subtilis and Haemophilus influenzae. However, most of them are pathogenic. Thus it is not possible work with them in a Biosafety level 1 laboratory.



Background:

Literature studies indicate that Acinetobacter baylyi ADP1 is a good chassis for our project because of its ability to degrade aromatic compounds and its naturally competency. It is non-pathogenic and belongs to risk group 1[1]. Recent work has been done to produce wax ester in Acinetobacter baylyi ADP1 [2]. Strains of Acinetobacter baylyi have been engineered that can utilize Gluconate and Glucose more efficiently than existing strains. [3]. However, a major shortcoming of this organism is that not many tools are available for genetic engineering. For example, typically only T5 and T7 are the two standard promoters used in engineering this organism.



Project:

Our objective was to create a T5 based synthetic promoter library for Acinetobacter baylyi ADP1. A reporter protein was required to measure the strength of promoter using fluorometry. We had approached GenScript for codon optimized GFP for Acinetobacter baylyi ADP1. Reliable codon usage table data for Acinetobacter baylyi was not available. . Hence, we made a free-to-use online tool called CUTE (codon usage table easy) that can generate a codon usage table by taking into consideration the genomic protein-coding annotation. This tool can be used for any organism whose coding regions are annotated in the genome. Cute can be found at cute.chassidex.org
Using this tool, we generated a codon usage table from the protein annotation data of Acinetobacter baylyi ADP1 which is available on the NCBI website. Using this table, we codon optimized GFP (which was codon optimized previously for E. coli).
Following this, we generated a T5 promoter-based library for Acinetobacter baylyi ADP1. Since these promoters are T5 based, they could potentially work in other gram-negative organisms like E. coli strains, Cornybacterium etc.


References:

  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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