Team:IIT-Madras/Description

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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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