# Multi-tiered containment system for engineered microbes

> **NIH NIH R01** · UNIVERSITY OF WISCONSIN-MADISON · 2022 · $305,672

## Abstract

The use of synthetic microbes to deliver therapeutics offers excellent potential to manage diseases in both
agriculture and human medicine. However, use of genetically modified organisms (GMOs) raises immediate
concerns with regards to their containment. Therefore, strategies must be developed that prevents permanent
colonization and accumulation of the GMO in the host and environment, respectively. Also, a kill-switch must be
in-place to eradicate the GMO efficiently. Especially in human medicine, an efficient kill-switch is needed to
overcome safety concerns when complications would occur as a consequence of an immune reaction against
the therapeutic molecule. Until we have overcome these fundamental technical challenges, clinical applications
of microbial therapeutics will be beyond reach in human medicine. Our long-term goal is to develop probiotic
bacteria as therapeutic delivery vehicles to improve human health. The overall objective of this application is to
establish containment and safety strategies which we envision will be broadly applicable in both gram-positive
and gram-negative bacteria. Our central hypotheses are a.) that deletion of genes encoding adhesins will reduce
the organism's ability to interact with host cells; b.) that thymidine auxotrophy will limit growth in the environment;
c.) that CRISPR-delivery by recombinant bacteriophages can specifically eradicate the target organism from a
complex environment. Our hypotheses have been formulated based on our published findings. We demonstrated
that L. reuteri adhesins are critical to adhere to epithelial cells, that cells lacking thyA are dependent on
exogenously added thymidine, that L. reuteri encodes biologically active bacteriophages in vivo, and that
CRISPR-Cas is functional in L. reuteri. The rationale for the proposed research is that successful completion of
this work is expected to yield a prototype of Lactobacillus reuteri that can be used for safe in situ delivery of
therapeutics to treat an array of diseases with an embedded biological containment system that is broadly
applicable in both Gram-negative and Gram-positive microbes. We plan to accomplish the overall objective by
completing three specific aims. In Aim #1 we will develop a delivery vehicle with reduced colonization potential.
In Aim #2 we will develop an environmental containment strategy based on thymidine auxotrophy. In Aim #3 we
will develop recombinant phages to achieve strain-specific killing of L. reuteri via CRISPR-Cas. L. reuteri phages
will be engineered to encode CRISPR arrays targeting engineered L. reuteri. Strategies will be implemented to
reduce `escapers', and in vivo efficacy and specificity will be determined. When successful, our work will have a
positive impact as we will have developed a functioning prototype of a safety strategy for a synthetic microbe,
which we expect will serve as a novel research tool to deliver therapeutics in situ. The evolutionary conservation
of thyA combined ...

## Key facts

- **NIH application ID:** 10335209
- **Project number:** 5R01GM135483-03
- **Recipient organization:** UNIVERSITY OF WISCONSIN-MADISON
- **Principal Investigator:** Jan-Peter van Pijkeren
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $305,672
- **Award type:** 5
- **Project period:** 2020-04-01 → 2024-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10335209

## Citation

> US National Institutes of Health, RePORTER application 10335209, Multi-tiered containment system for engineered microbes (5R01GM135483-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10335209. Licensed CC0.

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