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: 73%;"><strong>Motivations:</strong></span>
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<h4 align="left"><strong>Motivations:</strong></h4>
  
  
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<li>Initial Idea of lignin degradation to produce higher valued vanillin.
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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 look up the literature and we look at nature that can degrade the aromatic rings of Lignin. We needed an organism that could degrade 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 organism like <em>E.coli</em> and <em>L. lactis.</em>, we need to make competent cells for transformation. We looked up into literature and nature and found many ‘Naturally Competent’ microorganisms. However, most of them are pathogenic and known to cause harm to humans. Hence, you cannot work with it in 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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<span style="padding-right: 77%;"><strong>Solution:</strong></span>
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<h4 align="left">Background:</strong></h4>
  
 
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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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The organism that best fits into both requirements was <em>Acinetobacter baylyi</em> ADP1 strain. This organism is naturally competent, nonpathogenic, has amazing property of degrading aromatics. (the strain can be purchased from DSMZ.)
 
  
 
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<h4 align="left">Project:</strong></span></h4>
  
  
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<span style="padding-right: 73%;"><strong>Motivations:</strong></span>
 
  
 
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<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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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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We looked up the literature for <em>Acinetobacter baylyi</em> ADP1 but could not find substantial literature backing. The literature was too less and not many tools were available to do synthetic biology experiments with this organism. So, This was the motivation to generate a toolbox for <em>Acinetobacter baylyi</em> and try to make it as generic as possible so that if any other team or research groups wish to work with other organism then they can used these tools.
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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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<h4 align="left"><strong>References:</strong></h4>
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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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The first thing that is required for any synthetic biology experiments are fluorescent reporter proteins. We wished to codon optimize GFP and mCherry for <em>A. baylyi</em>. However,  when we asked the companies to codon optimize the fluoroscent proteins, the problem with companies was they did not have the codon bias table for <em>A. baylyi</em>.<br><br>
 
  
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<strong>Solution 1:</strong> We made a free use online tool called CUTE (codon usage table enumerator) that can generate Codon usage table by taking into consideration the protein coding annotation. This tool can be used for any other organism whose Genome has been sequences and protein coding regions are annotated. Cute can be found on the <a href="https://cute.chassidex.org" target="_blank">CUTE ChassiDex.</a><br>We generated the codon usage table as the protein annotation of <em>A. baylyi</em> is 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 its characterization can be found here. <br><br>
 
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<strong>Solution 2:</strong> Next we move 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 can have varying strengths. This library can be extremely useful for metabolic engineering and synthetic biology experiments.<br>
 
  
  
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Latest revision as of 15:09, 6 December 2018

iGEM Collaborations Page

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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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