Engineering transcriptional logic within bacteria using complex, modular, CRISPRi circuit arrays
Open Access
- Author:
- Roggenbaum, Morgan
- Area of Honors:
- Biological Engineering
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Howard M Salis, Thesis Supervisor
Ali Demirci, Thesis Honors Advisor - Keywords:
- synthetic biology
genetic circuits
CRISPRi
transcriptional read-through - Abstract:
- A key focus of synthetic biology involves engineering computation within living organisms. To accomplish this, biological response mechanisms are redesigned into synthetic gene networks. By implementing genetic circuitry, engineered cells can sense their environment and respond according to pattern recognition and pre-programmed logic. Such biotechnologies could be utilized in autonomous metabolism or disease control applications. Genetic circuits of limited complexity have been demonstrated in both prokaryotic and eukaryotic cells, however few are constructed using novel CRISPRi technology. The design and implementation of synthetic circuits is currently limited due to challenges in regulator cross talk, genetic part repetition, unpredictable genetic context, transcriptional read-through, and regulator balancing. If applied to circuit functions, CRISPRi has the potential to circumvent select synthetic circuit challenges. In this thesis work, two CRISPRi circuit obstacles were addressed including transcriptional read-through and regulator leakiness and balancing. To investigate transcriptional read-through, ten CRISPRi-based NOT gate cascade circuits were designed and constructed in E. coli, five featuring single terminators between sgRNA regulators (collection v1) and five featuring double terminators between sgRNAs (collection v2). Performance of the two circuit collections was analyzed and compared to examine the impact of termination efficiency on circuit function by way of reducing transcriptional read-through. Increased termination efficiency between sgRNA regulators improved sgRNA insulation and refined intended circuit signaling. With this phenomenon established, two additional circuit collections, v3 and v3.1, were designed next to address the challenge of regulator leakiness and balancing. Future work will include construction and characterization the v3 and v3.1 circuit systems. In completing this work, we are substantiating foundational rules for engineering CRISPR-based genetic circuits and producing a simple, scalable, robust method for constructing bacterial circuitry.