Human Practice

Alistair McCormick is the lead author and researcher behind the MoClo System and the toolbox to engineer the cyanobacteria PCC 11901. During our interview with Alistair McCormick, professor in Plant Engineering Biology at University of Edinburgh, we explored various facets of our research on cyanobacteria transformation and CRISPR techniques. We discussed the timeline for progressing from Level 0 vectors to Level 1, typically taking about a week, and compared integrative and self-replicative systems. Professor McCormick suggested that using E. Coli could expedite our processes, potentially eliminating the need for tri- and bi-parental mating. However, he noted that their method only involved one helper strain. He also pointed out the downsides of conjugation, such as the uncertainty of E. Coli dying on the plate, the need for constant re-streaking, and the importance of maintaining optimal growing conditions, could make the process tedious. Additionally, he mentioned that Synchococcus 6803 is a viable strain but not the fastest-growing or most well-studied strain; he suggested that we look into PCC 11901 as an alternative strain. Regarding construct generation, Professor McCormick detailed the process for creating Cas12a constructs and suggested that increasing the incubation period for cultures could improve efficiency from 25% to 80%. For level 0 parts, he emphasized the importance of careful mixing, labeling, and database management. He advised us to order or PCR it depending on the Level 0 requirements and being cautious with the primer design. He also recommended trying different assembly protocols if the initial one was to fail. For the guide design he suggested using tools like CasOT to analyze whole-genome FASTA files and SnapGene for zooming into potential guide RNA locations.

Given the importance of utilizing the MoClo and CyanoGate kits in our project, we reached out to Dr. Ravendran Vasudevan, the first author of the MoClo paper and a professor in the Department of Plant Sciences at the University of Cambridge. ^[7] With his expertise on the MoClo protocol, we sought his advice on refining our approach and optimizing the use of the MoClo kit. During our discussion, we mentioned using electroporation to further accelerate our transformation process. Dr. Vasudevan noted that while electroporation can be an effective method, its efficiency varies depending on the strain being used. During our discussion, we also compared the efficiency of self-replicating plasmids versus integrative plasmids. He highlighted that self-replicating plasmids integrate much faster, taking approximately 2 weeks compared to 3-4 months for integrative plasmids. He also referred us to his colleague, Alistair McCormick, for further questions on working with cyanobacteria.

One of our most impactful interviews was one with Shan Huang, a postdoctoral scholar in the Department of Pediatrics at Stanford School of Medicine. Dr. Shan Huang provided us with a deeper understanding of the complexities involved in utilizing Argonautes for guide DNA incorporation and the nuances of homology-based recombination. His expertise illuminated key aspects of our project, such as the implications of long homology sequences on system performance and the role of dead CbAgo in facilitating and encouraging recombination. Dr. Huang's guidance helped clarify how to optimize our experimental setup and anticipate potential challenges, ultimately strengthening our approach and refining our research strategy. ^[9] All this consolidated our understanding of our experiment and was overall helpful in attaining our goal.

Max Schubert is an expert working with cyanobacteria in hopes of helping climate change. He specifically uses Retron-Library Recombineering in order to study cyanobacteria. He was also researching similar systems to Lambda-Red Recombineering in cyanobacteria and we were curious if he had found anything. At that time our goal was developing single stranded guide DNA’s for our Argonaute Accelerator. As such we began looking into linearized DNA transformation techniques such as Lambda-Red Phage Recombineering for E. Coli and stumbled across a project proposal on genetic tools for photosynthetic microbes. During our meeting, Max Schubert shared his expertise on Retron-Library Recombineering and its potential use for gDNA generation. He was also ever so kind to offer to give us a couple cyanobacteria strains that he was working with, including 3154, which ended up the main focus of our project.

One of the people we interviewed was Susan Golden, a director of the Center for Circadian Biology and a Chancellor's Associates Chair (III) in Molecular Biology at University of California, San Diego. Our interview mostly focused on cyanobacteria incubation and culture growth in BG-11 media. During the interview, we seeked advice on our novel argonaute-accelerated cyanobacteria transformation protocol. In the interview, Dr. Golden explained how BG-11 is common in labs for cyanobacteria culturing as it contains nitrogen and other nutrients. She also emphasized the importance of incubation with fluorescent blue light for Synechococcus PCC 7942 and 2973 as both strains contain Phycocyanin. From here, we explained the fundamentals of our project, in which she pointed out her concerns regarding our experimental design. She mentioned that getting our payload into the cell should not be difficult for PCC 6803, as it is similar to PCC 7942 and the strains are naturally competent. The real challenge with these strains lies in achieving full penetrant integration. After the gene is successfully inserted, the cell must undergo homologous recombination to ensure that all copies of the chromosome carry the new gene. This process is crucial for stable gene expression but can be time-consuming and difficult to achieve, particularly in certain strains where homologous recombination is less efficient or consistent. ^[8] This step is critical for ensuring the success of genetic modifications, especially in applications where uniform gene expression across all cells is necessary. However, Dr. Golden also highlighted that eliminating the argonaute gene enhances transformation efficiency in cyanobacteria. Our experiments will clarify whether Argonaute systems are suitable for genetic engineering.

Realizing that much of our project would involve a lot of technical knowledge and skill working in spirulina we interviewed several experts on the matter. Hannah Tabakh (along with Ben) of Lumen bioscience gave us a lot of advice on engineering concerns in spirulina. They advised us to use GFP as a marker of transformation, they provided us with several techniques for aiding colony selection with these notoriously tricky mobile organisms and much more.

Realizing that much of our project would involve a lot of technical knowledge and skill working in spirulina we interviewed several experts on the matter. Ben Jester (along with Hannah) of Lumen bioscience gave us a lot of advice on engineering concerns in spirulina. He advised us to use GFP as a marker of transformation, he provided us with several techniques for aiding colony selection with these notoriously tricky mobile organisms and much more.

Lauren Goetsch is an associate scientist working with various biotechnological companies. Goetsch has a Bachelor of Science (B.S.) in Molecular Biology and has worked with the company focusing on transforming cyanobacteria, Lumen Biosciences. Her experience in working with cyanobacteria and Lumen Biosciences was sought out by us. We asked various questions on cultivating and transforming Limnospira Fusiformis (SAG85.79) and discussed our project. The talk with Goetsch helped us learn more about handling the strain as well as gain insight within our project.

We also spoke to ex Lumen employee Damien Carrieri. Damien explained to us that one of the greatest difficulties with engineering spirulina was a lack of good selectable markers and he helped us think about how to deal with this limitation. He also gave us advice on culturing spirulina and helped us brainstorm for how to check for the presence of active versions of the enzymes we were interested in producing in spirulina.