Background and motivation
B12 deficiency is framed as a solved problem in wealthy countries, just take a supplement. But in low resource environments where supplements are unavailable, unaffordable, or supply chains are unreliable, like my home country I recently visited as a vegan, this answer fails completely. B12 deficiency is already a serious public health issue in parts of South Asia and Sub Saharan Africa. Veganism and plant forward diets are effectively inaccessible without a reliable B12 source in these contexts.
The actual biological solution, gut bacteria producing B12 in situ, works fine for ruminants. It fails in humans because our synthesis happens below the ileal absorption site. The question I’m exploring: can we engineer around this?
I propose using Chassis: E. coli Nissle 1917, chosen for it’s established gut colonization ability, existing clinical probiotic use.
Delivery: Heat shock transformation to introduce Cas9 + gRNA + B12 operon construct. Plasmid carries CRISPR machinery for chromosomal integration at a safe harbor locus, then gets lost naturally post-editing, the impermanence of the plasmid becomes a feature, leaving only the stable chromosomal insert.
Why chromosomal integration rather than plasmid maintenance? Ther is no antibiotic selection pressure in vivo. Plasmid only approach loses expression within weeks.
Operon source: Aerobic B12 biosynthesis pathway from Propionibacterium freudenreichii. Full pathway is approximately 30 genes
I’m specifically asking whether a functional minimal subset has been characterized that would reduce integration complexity.
Wildtype Nissle 1917 produces no B12. Post-transformation, cobalt chloride supplementation feeds the pathway cofactor. Testing for methylcobalamin output confirms functional expression without requiring sequencing access. A microbiological assay using Lactobacillus delbrueckii as a B12 auxotroph provides a simple quantification method.
Has a minimal functional B12 operon been charconstruct fewer than 30 genes that still produces physiologically meaningful output?
What safe harbor loci are recommended for Nissle 1917 specifically? I’ve seen literature referencing lacZ and agaI in K-12 strains but Nissle’s genome differs.
Any published data on colonization persistence of chromosomally engineered Nissle strains in vivo?
Does anyone have experience with Addgene shipping to non-standard countries, or alternative sources for Cas9/gRNA constructs?
Most importantly, i would love some feedback on this project :3