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What is the context of our research?

Using the native sludge bacteria Nitrosospira multiformis and Nitrosomonas eutropha as chassis we inserted genes to produce erythromycin esterase B and β-lactamase to breakdown azythromycin and penicillin. We also inserted nirS, norB, norC, and nosZ from Pseudomonas aeruginosa PAO1 to convert nitrates into nitrogen gas, as well as genes to produce dispersin, amylase, and AHL-lactonase to inhibit the biofilm formation which blocks helpful bacteria from functioning fully. To increase bacteriophage resistance, prophage in the Nitrosospira and Nitrosomonas genomes were identified and used to build a guide RNA region for a Type II CRISPR system. These improvements will help reduce antibiotic resistance, increase water reclamation, prevent algal blooms, and allow more biomass to be harvested.

What is the significance of our project?

Wastewater facilities face challenges in effectively processing waste including residual antibiotics, excess nitrates, biofilm buildup and low survival rates of microbes essential to biodegradation. Our work will provide more effective solutions to handling these issues.

What are the goals of our project?

• Improve bacteriophage resistance 1000-fold in N. multiformis and characterize the CRISPR system allowing for this increased resistance. This will be the first time this particular CRISPR is being characterized.
• Introduce denitrification genes into N. multiformis to convert ammonia into nitrogen gas. This will allows us to remove excess nitrates from sewage effluent and prevent harmful environmental outcomes like eutrophication.
• Introduce genes into N. multiformis to degrade two of the most common classes of antibiotics found in wastewater.
• Test efficacy of genes that degrade biofilms that we have introduced into N. multiformis.
• Test genes that will eliminate our modified bacteria if they leave the wastewater (acts as a control).
• Present our research at the international iGEM jamboree!