HUMAN PRACTICES

Our discovery work converged on a project in environmental remediation. As a generation raised in the shadow of global warming, we understand that the effects of environmental crises are extensive, and that they disproportionately impact under-equipped communities. We wanted to model a project that demonstrated the potential for synthetic biology to effectively address environmental crises.

With this, we first considered remediation of one of the most prevalent toxins in marine ecosystems: domoic acid. Domoic acid is a potent neurotoxin released by Pseudo-nitzschia australis during HABs; the toxin seasonally contaminates marine ecosystems through accumulation in shellfish and sardines. Organisms that ingest contaminated fish are poisoned; the environment and economy suffer when toxic HABs persist. We recognized that the frequency of toxic HABs would only increase with global warming, so we wanted to address this with synthetic biology.

TABI began with a meeting with Professor Raphael Kudela at UCSC, who specializes in phytoplankton ecology. He referred us to multiple researchers that would have insight into how toxic HABs impact marine ecology. This meeting was also when we were introduced to Microcystis aeruginosa and microcystin contamination in Pinto Lake.

With Dr. Kudela's referral, we spoke to Monica Thrukall, a UCSD graduate student in the Allen Lab studying Pseudo-nitzschia australis. She informed us that domoic acid remediation was unfeasible because Pseudo-nitzschia australis blooms were too distributed, and techniques for engineering this species were under-developed. This is the first time we felt frustrated by the lack of generalized techniques for engineering non-model species. We began considering generalized solutions for this limitation, and we shifted our remediation efforts toward a more local environment: microcystin contamination in Pinto Lake.

We had the privilege of meeting with Bryan Condy, the laboratory manager for the City of Watsonville. We discussed issues faced by Pinto Lake, noting that there has been an increase in toxic HABs since the 1980s. Bryan informed us that the warming climate, chronic agricultural runoff, and excessive phosphorus levels from sediment at the bottom of Pinto Lake are the main cause of HABs. Representing a low-income community, Bryan stressed the need to preserve free, safe, and accessible outdoor opportunities in the under-served community of Watsonville.

Our effort towards microcystin remediation directed us to a meeting with Professor Shaun McKinnie of UCSC who specializes in biologically-significant natural products and their related enzymes. Dr. McKinnie introduced our team to the mcy gene cluster that synthesizes microcystin, and recommended research into degradation genes within the genomes of M. aeruginosa and its competitors. We decided against the expression of microcystin-degrading enzymes in M. aeruginosa because we wanted to address the source of the problem: microcystin production.

We decided to pursue targeted mutagenesis of a gene within the mcy cluster to selectively disrupt M. aeruginosa toxicity while maintaining cell viability. We believed this approach would work to remediate microcystin toxicity in Pinto Lake with minimal ecological disruption. We consulted with several researchers to help draft our protocol. Notably, Dr. Glenn Millhauser and Dr. Kevin Singewald of UCSC provided us with guidance about microcystin analysis, and Dr. Diego Gelsinger of Columbia University aided us in prokaryotic genome engineering.

We designed a plasmid capable of targeted mutagenesis of a gene within the mcy cluster. We were excited by the support we had with regards to genomic integration, but we recognized that this approach would only be viable if the plasmid was capable of being delivered by HGT.

Our objective shifted toward developing efficient methods for HGT into M. aeruginosa. Dr. Gelsinger supplied us with a conjugative strain of E. coli (EcGT2) that could conjugate with other species without requiring a helper plasmid. He informed us that conjugation efficiency was low in general, and that the process was difficult to optimize in the lab—much more so in the environment. It became clear that a generalized, bioinformatic approach aimed at improving transformation efficiency in non-model species was the most impactful contribution that TABI could provide to the iGEM community.

Our principal investigator, Dr. Bernick, introduced us to Stealth (section 5.1.1), a cutting-edge bioinformatic program written to enhance genetic engineering in non-model bacterial species like M. aeruginosa. Stealth facilitates an advanced statistical analysis of a specific organism's genome to identify underrepresented motifs that may have faced negative-selective pressure by targeting via endogenous restriction modification (R-M) systems. Resulting data allows for evasion of the host’s R-M system by virtue of pattern avoidance.

The M. aeruginosa strain we are working with, UTEX 2385, is a non-model organism. To use Stealth, we needed to sequence UTEX 2385’s genome. We completed the first round of sequencing using the Oxford Nanopore, but the genome was partially sequenced and the sample included DNA from organisms besides M aeruginosa. We then reached out to Brandy McNulty, a Nanopore Production Specialist at the UC Santa Cruz Genomics Institute. She helped us troubleshoot our sequencing protocol and provided guidance throughout our sequencing runs.