About This Project
Exciting new approaches use bacteria to capture carbon, mitigate fertilizer use, or other climate interventions. However, Bacteria are not well-adapted to these new-to-nature conditions, and many such technologies could be improved by domesticating bacteria to improve their performance and climate impact. BioBloom is a new method of barcoded directed evolution at the genome-scale, and this project pilots this method in a fertilizer-producing bacterium and a fast-growing photosynthetic bacterium.
Ask the Scientists
Join The DiscussionWhat is the context of this research?
For the first time, it’s possible to measure the phenotype of all possible mutations in a genome. This is due to advances in DNA sequencing and synthesis, and to new techniques enabling multiplexed experimentation, in which each cell can perform an experiment and produce data. The previously-developed Retron Library Recombineering(RLR) technology, measures the effect of millions of mutations in bacteria. In contrast with CRISPR, RLR scales to millions of library members because of it’s cut-free mechanism. In Phase I, the project de-risked Soft-randomized-RLR toward barcoded, saturating mutagenesis of bacteria. Next, adapting this method to both fertilizer-producing and carbon-fixing bacteria can "domesticate" them for GHG mitigation and removal.
What is the significance of this project?
This project will develop a new tool for directed evolution of bacteria being used for fertilizer replacement and CO2 utilization. Compared to laboratory evolution, the BioBloom approach i) is faster, ii) is comprehensive, testing a known set of mutations, iii) produces quantitative phenotypic data for millions of mutations, ideal for training models driving further advances.
Improved genetic tools help multiple bacterial approaches to make impact. Air Protein, Solar Foods, Circe, Lanzatech, Pivot Bio, Kula Bio, and Switch Bioworks use bacteria to fix CO2 and N2 to produce food and replace fertilizer. BioBloom aims to evolve bacteria underlying these approaches for improved growth and performance.
What are the goals of the project?
In Phase I BioBloom de-risked that Soft-randomized RLR can create random barcoded libraries covering the genome of E. coli. The next step: adapt this to climate-relevant bacteria.
K. variicola (Kv) is an N2-fixing E. coli-relative used as a fertilizer replacement on millions of acres. Exonuclease KO and expression constructs will be tested to develop efficient RLR in Kv. If successful, this approach can help generate Kv making a bigger impact on fertilizer, and could be a platform for sequestering carbon in soils as well.
I will also test X. autotrophicus, an N2-fixing bacterium used to make food from CO2. Less is known about RLR editing in this bacterium, so I will also need to test both synthesized SSAP protein variants and exonuclease KO.
Budget
These budget items support the proposed research plan, including materials to grow the climate-relevant bacteria, edit their genomes, and test the Biobloom approach in these bacteria. Currently BioBloom is being tested in E. coli only using external funding.
Adapting the BioBloom approach to these new bacteria requires obtaining, growing, and banking these new bacteria, and the media and labware help support this. In addition, DNA constructs for retron editing and potentially for knockout of exonucleases must be constructed and delivered to these hosts, which these support.
A substantial technical risk for BioBloom functioning in cyanobacteria is having an efficient SSAP, which has never been demonstrated in these organisms. 21 total potential SSAPs gleaned from protein databases or through bioinformatic mining of metagenomes in Phase I will be synthesized, constructed, and tested using an NGS pipeline.
Partial salary and rent support is requested to supplement existing funding.
Project Timeline
In Phase I, K. variicola (Kv) was obtained and and successfully transformed. Next exonuclease knockouts (exo KOs) will be created, and RLR editing tested in Kv. Different vectors and expression levels may also be tested, with expectation for a working system by April 2025.
In Xanthobacter autotrophicus (Xa), I will construct exo KOs as well, but also need to synthesize and test different SSAP proteins. I expect to complete this de-risking work by April 2025.
Dec 31, 2024
Demonstrate conjugation in Xa
Dec 31, 2024
Demonstrate conjugation in Kv
Jan 31, 2025
Synthesize and clone 21 SSAP variants for Xa
Jan 31, 2025
Construct exo KOs in Xa
Jan 31, 2025
Construct exo KOs in Kv
Meet the Team
Affiliates
Max Schubert
After Double majors in Microbiology and Molecular Environmental Biology at UC Berkeley, Max was introduced to the world of Synthetic Biology through his work at Amyris, where he contributed to the development of CRISPR methods and product development.
Max then began PhD studies at Harvard University, completing a thesis in the lab of George Church. His thesis work focuses on Retron Library Recombineering, a new approach for pooled screening in bacteria, alongside contributions to other studies and helping organize the Boston Bacterial Meeting conference.
Wanting to work more explicitly in climate technology, Max then began work in photosynthetic bacteria, helping to develop automated culture systems for cyanobacteria, directed evolution approaches for cyanobacteria, and contributing to studies on genome streamlining of cyanobacteria. This work includes a bio-prospecting study in which novel fast-growing cyanobacteria were isolated from shallow, marine CO2-emitting volcanic seeps. These bacteria were found to possess traits promising for carbon-sequestration, and are now being investigated for this purpose.
Max gained broad exposure to the world of climate technology through his role as a Scientific Advisor to the Grantham Foundation for Protection of the Environment, and serves as a science advisor for two small climate start-ups.
The BioBloom project resulted from realizing that many climate efforts needed a technology that is unavailable, but potentially related to Max's thesis work on genetic methods. Max is now full-time on BioBloom, and believes this is the best way he can make a climate impact!
Lab Notes
Nothing posted yet.
Additional Information
Domesticating bacteria can be relevant to GHG mitigation and removal in multiple ways. The bacteria used here are chosen strategically both because of the tractability of getting RLR to work, and because of their multiple avenues of climate impact.
Klebsiella variicola (Kv) is one of the most abundant members of the root biomes of many crop plants, and can be used to partially replace nitrogen fertilizer in crops (1,2). Further, that this engineered bacterium can use the energy from crop plants (directed through their root exudates) to perform a costly function designed by humans may indicate it could be a platform for performing other functions, like carbon sequestration. This approach is especially interesting because it uses already managed ecosystems (agriculture) to perform an ecosystem service without new built infrastructure or energy input.
Xanthobacter autotrophicus (Xa) is a less well-understood bacterium, but has been shown to be useful for fixing both Carbon and Nitrogen, for manufacturing food, food products, or fertilizer. Companies known to use this bacterium include Solar Foods, Kula Bio, Air Protein, and others. By supplying energy from electricity in the form of Hydrogen gas, Xa growing in a fermentor fixes both CO2 and N2 in order to manufacture products. Xa is also one of the bacteria most capable of growing on formate, one of the easiest compounds to create electrochemically from CO2, but often quite toxic. This could be an alternative strategy to feeding hydrogen/CO2, overcoming some of the scaling/solubility issues of these strategies when thinking about utilizing CO2 to make an even wider range of things. Xa is known to make biodegradable bio-plastics, which could be used to replace petrochemical plastics, and create carbon-negative materials.
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