Engineering

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Limnospira Fusiformis is a difficult to transform cyanobacteria known for its nutritional content, which our team hoped to leverage to produce ingredients of premium baby formula. Even as we pivoted to differing strains such as UTEX 2973, 3154, and PCC 7942, the problem of transforming polyploid cyanobacteria proved difficult and time-intensive to solve. To make polyploid cyanobacteria genetically tractable, we needed to overcome the inherent Restriction Modification systems. To overcome this immune system, we developed BLACKBIRD to utilize the STEALTH algorithm and remove possible RMs targetted sights from our gene insert as well adapting the insert to the target’s codon bias. Below we detail the Design-Build-Test-Learn process we undertook on the program to ensure its viability in engineering.

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Design-Build-Test-Learn cycle

The BLACKBIRD version 'Alpha' has achieved notable success in designing custom inserts. The program processes an insert sequence and refines it through multiple iterations of STEALTH, aiming to reduce the number of recognition sites utilized by the target organism's native restriction enzymes.

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BLACKBIRD is a versatile program that can be calibrated for different organisms using a complete strain-specific genome in FASTA format and a STEALTH software output. The STEALTH [1] output file contains the k-mers that correspond to hypothetical RM recognition sites; the generated k-mers will be replaced from the custom insert. During the initial development of BLACKBIRD, we utilized the genome and STEALTH sites generated for the well-documented UTEX 2973 strain. Using these specifications, we customized our three potential inserts accordingly: GFP, CbAgo and Cas12a.


BLACKBIRD Process

This process was then replicated for UTEX 3154, our target organism. Since our target organism's genome has not been officially sequenced, BLACKBIRD currently operates using the PCC 11901 genome, which is closely related to UTEX 3154. The outcomes of BLACKBIRD for both UTEX 2973 and PCC 11901 are shown in the bar graph.

The number of STEALTH hits after a comparable number of iterations through the program for inserts customized to PCC11901 seem to still contain almost 30% of their initial number of hits whereas that number is closer to 5% when customized to UTEX 2973. Within the current version of BLACKBIRD, this result can be accounted for by the fact that the number of STEALTH hits for UTEX2973 and PCC11901 are 283 and 754 respectively.


The STEALTH list of k-mers are generated based on a user-determined cut-off value. We initially ran BLACKBIRD on a False Discovery Rate score (or bootstrap score) of around 72 as our cut-off. Upon discovering the unreliability of the results, this bootstrap score was raised to 86. Because it is less lenient when generating the list of k-mers, the overall conditions make for better BLACKBIRD results. The right graph in figure shows the results of moving this cut-off; in one example, we were able to reduce the number of hits of the PCC11901 customized GFP from 41 to a mere 6. This was a very predictable result due to the fact that the number of hits for the higher bootstrap score was similar to that of UTEX2973 for all inserts due to the fact that the number of STEALTH hits for both conditions was both around 280.

References

[1]S. Hu, S. Giacopazzi, R. Modlin, K. Karplus, D. L. Bernick, and K. M. Ottemann, “Altering under-represented DNA sequences elevates bacterial transformation efficiency,” mBio, vol. 14, no. 6, Oct. 2023, doi: https://doi.org/10.1128/mbio.02105-23.

[2]G. Zhang, "Transient ribosomal attenuation coordinates protein synthesis and co-translational folding" Nat Struct Mol Biol, Jul. 13, 2008 https://www.nature.com/articles/nsmb.1554 (accessed Sep. 26, 2024)

[3]G. L. Rosano, "Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted Escherichia coli strain" Microb Cell Fact, Jul. 24, 2009 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2723077/ (accessed Sep. 26, 2024).