Progress 10/01/23 to 09/30/24

Outputs
Target Audience:The target audience includes stakeholders in academia who are interested in research on synthetic biology, biomolecular engineering, biological engineering, and bioconfinement; policy makers who regulate the application of recombinant microorganisms for environmental remediation and other biotechnology purposes; as well as industries and other stakeholders who are interested in developing recombinant microorganisms for various biotechnology applications. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One Post-doc and one undergraduate student in Biological Systems Engineering were trained for metabolic engineering and synthetic biology. The Post-doc is preparing a manuscript for publication based on the results out of this research. How have the results been disseminated to communities of interest?We have presented the relevant project concept and research results during various presentations, including several invited talks by the PD. The PD has also disseminated the project concept and relevant results through guest lectures, seminars, and various teaching activities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? This project sought to create robust light-inducible suicide circuits (light-inducible, LISC and darkness-inducible, DISC). The broader vision involves deploying engineered microorganisms in applications such as environmental remediation and bioproduction, where controlled containment is essential. Over the course of the project, we constructed, tested, and iterated on various regulatory designs to reduce leaky expression and enhance system stability. Alongside these suicide modules, a degradation pathway for 1,2,3-trichloropropane (TCP) was introduced to illustrate potential real-world utility. The outcomes demonstrate significant progress in engineering light-triggered containment systems with promising safety and reliability features. 1) Develop a suicide circuit with multiple antibacterial mechanisms. We initially tested endonuclease approaches such as EcoRI, Cas9, and Cas12 to induce DNA damage. EcoRI clones were unstable, showing frameshift or nonsense mutations, but co-expression of Cas9 (targeting the essential 16S rRNA gene) and mfLon (a protease degrading MurC) proved highly lethal, with over 99% killing under induced conditions. This multi-layered approach provided a strong foundation for subsequent constructs, underscoring the advantage of combining different lethal strategies. 2) Fuse a pdt tag to the end of murC. To enable mfLon protease to degrade MurC, we used CRISPR-Cas9 to fuse a "pdt" tag at the 3′ end of murC in an envZ-deleted strain (ΔenvZ). The resultant strain (ΔenvZ::pdt) displayed a 99.5% kill rate upon mfLon induction. This step validated the protease-based suicide mechanism and ensured robust control of bacterial viability via targeted protein degradation. 3) Delete the native lacI gene in the host strain. To avoid interference with our synthetic LacI-based regulatory circuits, we removed the native lacI gene from the ΔenvZ::pdt strain. The resulting host (ΔenvZΔlacI::pdt) served as the primary chassis for LISC development, reducing background expression that could compromise dynamic range or circuit stability. 4) Evaluate LISC in ΔenvZΔlacI::pdt. We split the LISC design across three plasmids: one carrying the light sensor (Cph8, plus genes for phycocyanobilin biosynthesis), another containing regulatory elements (OmpR, LacI, T7 RNA polymerase), and a third harboring the effector modules (Cas9 + 16S rRNA-sgRNA, mfLon). Different promoter and ribosome binding site (RBS) strengths were tested to control expression levels. This modular strategy provided the flexibility to fine-tune both the light response and the lethal output. 5) Improve the dynamic range by engineering the host strain. After knocking out envZ and lacI, we removed ompR to prevent extra copies of OmpR from causing background expression. The resulting strain (ΔZIRXCF::pdt) displayed a 48.4-fold improvement in GFP output when comparing dark and red-light conditions, highlighting the importance of removing genomic regulators that interfere with synthetic pathways. 6) Construction of light-induced GFP indicator and LISC circuits. We replaced gfp with lacI in various constructs under T7 RNA polymerase control. The aim was to repress T7-driven gene expression in the dark while allowing induction under red light. Although one variant achieved a moderate 1.4-fold change in GFP, leaky expression persisted in some designs, limiting overall induction ratios. These findings guided subsequent re-engineering to minimize background transcription. 7) Introduce dCpf1 for feedback inhibition of lacI. In an effort to reduce LacI "leakage," we introduced a catalytically inactive dAsCpf1 under red-light conditions. The plan was for dAsCpf1 to bind to lacI via a constitutively transcribed crRNA, blocking residual transcription. However, while this feedback mechanism lowered leakiness slightly, it did not fully resolve the challenge of background LacI expression. 8) DISC construction and cell suicide testing. We constructed a dark-inducible suicide circuit (DISC) by replacing gfp with mfLon. Under dark conditions, 57 -72.5% of cells were killed, demonstrating partial success in controlling lethality. However, stronger T7 promoters caused 100% cell death even under "off" conditions, indicating excessive leakiness. Balancing promoter strength and leak reduction remained a major design hurdle. 9) Genomic integration of suicide mechanism genes. To enhance both stability and control, we integrated the phycocyanobilin pathway and Cph8 sensor genes into the chromosome, followed by Cas9-sgRNA and mfLon. This genomic version of DISC achieved 89.2 -99.3% killing, surpassing plasmid-based counterparts. The results confirm that stable chromosomal integration can improve reliability and minimize plasmid-related variability. 10) Select an alternative light-control system. The Cph8 -OmpR circuit exhibited high background expression and lower-than-desired induction capacity, especially when driving potent killing cassettes. We pivoted to single-component or blue-light systems, aiming for tighter control and reduced genetic load. This shift sought to simplify the regulatory network and avoid multi-step signal transduction that can amplify noise or lead to unpredictability. 11) Protein modification and genome editing to eliminate native interference. In adapting the new light regulators, we removed interfering endogenous genes in E. coli to maintain signal specificity. Double-deletion strains further reduced cross-talk and formed a clean background for evaluating these novel single-component light-control systems. 12) RBS and promoter engineering for tighter regulation. We tested five RBS variants (RBS-a to RBS-e) and a panel of J23 synthetic promoters. Stronger expression sometimes reduced leakage but limited maximum induction, while weaker promoters allowed higher induced output but also higher background. RBS-c provided a good balance, achieving a favorable ratio between the "on" and "off" states. 13) Engineering operator sequences for lower leakage. We inserted additional operator sites near the -10 or -35 region of the promoter to further tighten repression. While one configuration severely reduced total expression, two variants showed minimal leakage alongside adequate induced expression. This step highlighted how subtle DNA architecture changes can profoundly affect circuit performance. 14). Construction of TCP degradation pathway. To demonstrate a practical application, we introduced a robust TCP degradation pathway into our engineered strains. Cells expressing these genes could tolerate and fully degrade up to 1.5 g/L TCP within 40 hours, overcoming growth inhibition in higher TCP concentrations. This result underscores the potential for coupling biocontainment strategies to real-world bioremediation tasks. 15) Test cas12 + sgRNA killing system. We evaluated the miniaturized Cas12 system on plasmids, observing effective cell killing but varying efficacy depending on sgRNA choice. Proper copy-number control was essential to minimize leakiness. These findings establish a blueprint for harnessing Cas12 in multi-copy or single-copy contexts. 16) Integration of light-regulating operator and cas12 into the genome. Employing transposon-based gene editing, we integrated one to six copies of the cas12 cassette into different loci (e.g., gap, lacZ, acrR) of the E. coli genome. After excising the transposon, we obtained clean mutants with copy numbers ranging from 1 to 6. This approach provided a range of expression levels for tuning the balance between leak control and kill efficiency. 17) Expression of light-regulating proteins and sgRNA for a complete system. Finally, we introduced plasmids carrying the new light regulator and sgRNA cassettes into strains harboring the genome-integrated cas12 modules. This setup is designed to allow precise, light-dependent activation of the killing mechanism.

Publications