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Silver Medal Criterion #1

Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the Registry for your team to be eligible for this criteria.


Please see the 2023 Medals Page for more information.

Introduction

Microcystis aeruginosa is a naturally competent, non-model cyanobacteria that is principally responsible for toxic HABs in freshwater ecosystems. Our team sought to leverage natural competence to selectively transform M. aeruginosa in a complex bacterial community. Initially, we designed a plasmid that would disrupt the production of microcystin in transformed cells. However, to disrupt microcystin production at the scale of a HAB, we recognized a greater need to optimize the transformation efficiency in non-model organisms like M. aeruginosa. Low transformation efficiencies in non-model organisms can partially be attributed to the targeting of foreign DNA by restriction-modification (R-M) systems. R-M systems degrade foreign DNA by targeting short, specific nucleotide sequences with restriction enzymes. We believed that transformation efficiency in non-model organisms could be improved by avoiding R-M motifs in plasmid synthesis. To this end, we developed the Chameleon program. Our work is built off of the Stealth program, which produces putative R-M motifs by identifying short nucleotide sequences that are underrepresented in a specific organism’s genome. Chameleon removes putative R-M motifs from protein-coding regions of synthetic plasmids through synonymous codon-optimization. The following DBTL cycles were aimed at validating the use of Chameleon in future bioengineering projects by natural transformation of M. aeruginosa with modified and unmodified plasmids.

Cycle 1 - Transformation of M. aeruginosa

Design

M. aeruginosa is known to be naturally competent [1], but our team needed to devise a protocol for quantifying transformation efficiency. For this purpose, we ordered pSHDY (addgene#: 137661): a broad-host range, high-copy plasmid that's been engineered for expression in cyanobacteria.

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Cycle 2 - Genomic Sequencing

Design

The Stealth program identifies putative R-M motifs by determining short nucleotide sequences that are underrepresented in a specific organism's genome. To use Stealth on M. aeruginosa, we had to sequence the genome of our specific strain (UTEX 2385). Our team decided to use minION nanopore sequencing [2] because our project depended on rapid genome assembly, and it's much easier to assemble long reads produced by nanopore.

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Cycle 3 - Chameleon Validation

Design

The pSHDY backbone (Fig.1) was used to generate modified and unmodified pSPDY constructs (Fig. 2) using Golden Gate Assembly. In addition to the pSHDY backbone, pSPDY contains an enhanced GFP gene, a RP4 mobilization gene, and origin of transfer for end-point conjugation from EcGT2 E. coli to M. aeruginosa. The unmodified pSPDY construct retains Stealth-identified R-M motifs, while the modified pSPDY construct has been passed through Chameleon. Chameleon removed palindromic, Stealth-identified R-M motifs from protein-coding sequences by synonymous codon-optimization.

The pSPDY plasmid includes an eGFP gene to report activity of differential ribosome binding sites (RBS). We will synthesize two modified pSPDY constructs with different RBS associated with the eGFP gene: one will include an E. coli-derived RBS, and the other will includ a putative M. aeruginosa RBS. The putative M. aeruginiosa RBS was identified bioinformatically from nucleotide sequences directly upstream of the M. aeruginosa PCC 7806 ribosome genes, and verified by the 16S rRNA anti-RBS sequence. Thus, expression of eGFP will allow for validation of the putative M. aeruginosa RBS. With this information, we will be able to contribute a potentially-useful RBS for cyanobacteria envineering to the iGEM community.

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Cycle 4 - M. aeruginosa Ribosome-Binding Site Verification

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