The genetic toolkit at our disposal is rapidly expanding, yet the application of such tools is greatly limited to a small set of genetically tractable model organisms. Overcoming the barriers to genetic tractability in non-model organisms will advance the potential for synthetic biology to address real-world problems. The presence of Restriction-Modification R-M systems is widespread in prokaryotes and presents one of the most significant barriers impeding genetic tractability in non-model organisms [1].
These barriers are currently addressed by Ad hoc, organism-specific solutions which include mimicry-by-methylation, restriction site avoidance, and high throughput transformation of plasmid DNA [1]. These processes are restrictive in that they require a well-characterized restriction-modification (R-M) system, which are lacking in non-model organisms, or demand expensive and often unrealistic resource allocation [2]. This reality restrains the scope of innovative and novel engineering endeavors, hindering the perfusion of real-world syn-bio soltions that should be possible with well charecterized technologies. This barrier is especially evident in Microcystis aeruginosa, which possesses an extensive and robust R-M system that is largely uncharacterized [3]. Our team recognized that effectively engineering M. aeruginosa would require careful consideration of its R-M system, which is known to be strain-specific. This barrier was particularly frustrating in the context of a time-constrained iGEM project.
This foundational requirement of our engineering approach needed to be addressed, and we felt that the greatest contribution that TABI could make to the iGEM community would be a generalizable solution to restriction site avoidance. We developed the Chameleon project with the motivation to provide an automated platform for future iGEM teams to more effectively engineer non-model species.
| Part Number | Part Name | Part Use |
|---|---|---|
| BBa_K4592000 | CaMV35S Core Promoter | Short-length constitutive core promoter for use with cyanobacteria, algae, plant, and other photosynthetic chassis; also functional in other organisms (e.g. E. coli). The short length of the CaMV35S core promoter was leveraged to help avoid unremovable restriction sites on our plasmids. The CaMV35S core promoter reduces the size of the original promoter from 343 base pairs to 91 base pairs. |
| BBa_K4592001 | Microcystis-optimized eGFP CDS | Synonymous sequence to the eGFP gene that is codon-optimized for expression in UTEX 2385 M. aeruginosa and has had putative restriction sites identified from UTEX 2385 M. aeruginosa. The synonymous sequence was produced by processing of the original eGFP gene via the Chameleon project. The synonymous sequence is proposed to be more efficiently expressible and transformable in UTEX 2385 M. aeruginosa due to the codon-optimization and restriction site removal, respectively, though this has not been empirically validated yet. |
| BBa_K4592002 | Microcystis-optimized Cyanobacteria Reporter | Composite reporter built from BBa_K4592000 and BBa_K4592001 to express eGFP in UTEX 2385 M. aeruginosa. The composite reporter to form an insert into a plasmid to validate respective component parts in UTEX 2385 M.aeruginosa transformants. |
| BBa_K4592003 | Synthetic RBS for Microcystis-optimized eGFP Expression | High predicted translation initiation rate synthetic RBS designed with BBa_K4592001 as target protein CDS and PCC 7806 M.aeruginosa as target organism using Salis RBS Library Calculator v2.1.1. The synthetic RBS was designed to be used in a future composite part based on BBa_K4592002 in order to further improve expression in UTEX 2385 M.aeruginosa. The PCC 7806 strain was used as the target organism rather than the UTEX 2385 strain as it was the closest available, however the predicted features are expected to be similar due to the strains' similar anti-Shine-Dalgarno sequences that would interact with the RBS. |
References
[1] Q. Yan and S. S. Fong, “Challenges and Advances for Genetic Engineering of Non-model Bacteria and Uses in Consolidated Bioprocessing,” Front. Microbiol., vol. 8, p. 2060, Oct. 2017, doi: 10.3389/fmicb.2017.02060.
[2] S. M. Brooks and H. S. Alper, “Applications, challenges, and needs for employing synthetic biology beyond the lab,” Nat. Commun., vol. 12, no. 1, p. 1390, Mar. 2021, doi: 10.1038/s41467-021-21740-0.
[3] L. Zhao, Y. Song, L. Li, N. Gan, J. J. Brand, and L. Song, “The highly heterogeneous methylated genomes and diverse restriction-modification systems of bloom-forming Microcystis,” Harmful Algae, vol. 75, pp. 87–93, May 2018, doi: 10.1016/j.hal.2018.04.005.
[4] S. Hu, S. Giacopazzi, R. Giacopazzi, K. Karplus, D. Bernick, and K. Ottemann, “Altering under-represented DNA sequences elevates bacterial transformation efficiency.” University of California, Santa Cruz, Aug. 06, 2023.