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MotifFinder
MotifFinder
What if we could find de novo motifs in the diatom genome through computational methods? Then, we could mine the genome for transcription factor binding sites which we could then utilize in our synthetic promoters!
The above project is a command line utility written in Rust, the programming language, to find such de novo motifs in any genome. It allows for a unique strategy of Randomized Motif Search and Gibbs Sampler to be used to find the best set of motifs across the genome.
By compiling a list of 500bp upstream sequences of all gene start sites, we can create a file that can be used by MotifFinder to identify de novo motifs as well as search for known motifs such as the TATA box.
Construct Development
Construct Development
Using molecular cloning techniques (PCR, 3G assembly), the plasmid was constructed containing constitutive expression systems. The plasmid contained various essential components including CENS-ARS-HIS, the artificial yeast centromere piece which would allow for transformation of the vector into P. tricornutum.
After each step, verification of assembled constructs was performed to ensure adequate use downstream.
Transformation into dh5-α
Transformation into dh5-α
In order to test the efficiency of our promoter system, we transformed the vector created into dh5-alpha, a strain of E. coli. This resulted in relatively successful transformations which could allow for subsequent transformation into P. tricornutum.
Future steps
Future steps
With successful reproduction of the vector with the CENS-ARS-HIS component, we can then transform it into diatoms. We can verify the strength of each of the promoter systems using the GFP expression. Future directions could include functional genes that could produce something useful to test the efficacy of the system and the diatom's ability to create proteins.
Nithish Narasimman
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