Sequencing guided genetic part engineering

  • Matthew J Tarnowski

Student thesis: Doctoral ThesisDoctor of Philosophy (PhD)


Microorganisms shape the living world through activities encoded in their DNA sequences. Synthetic biology often involves modifying the behaviour of microorganisms with designed DNA made up of genetic parts to achieve complex biological functionality. Large libraries of genetic designs can be constructed using DNA assembly and amplification, which could enable a broader understanding of how to design sequences encoding specific functions. However, methods to characterise sequence and function are often limited in scalability and scope since they can only characterise individual genetic parts. DNA and RNA sequencing open new possibilities for characterising entire DNA libraries. Here, we apply nanopore sequencing, a technology where DNA sequences are read as they pass through nanoscale pores (nanopores), to study assembled libraries of genetic parts. DNA sequencing shows that one-pot combinatorial DNA assembly by ligation reliably constructs libraries from multiple genetic parts and that library composition is significantly more uniform when DNA is amplified without protein expression. The libraries encode transcriptional terminator genetic parts that signal where transcribing RNA polymerases (RNAPs) should dissociate from DNA. We use terminators as ‘transcriptional valves’ to tune RNAP read-through and control transcript isoform abundance, offering a new mechanism to regulate genetic designs transcriptionally. We develop a method to characterise the in vitro transcription of valve libraries simultaneously at nucleotide resolution using nanopore direct RNA sequencing (dRNA-seq). This method reveals that upstream sequence context changes how much termination occurs, along with genetic design principles for tuning and insulating terminator function. Using this knowledge, we then engineer an array of CRISPR guide RNAs transcriptionally regulated by our valves. With DNA being the code of life, synthetic biology comes with a responsibility to the living planet. Therefore, a framework for responsible innovation is used to assess potential real-world impacts of the arrays. This work provides new avenues for studying DNA library composition, regulating transcription and innovating biotechnology responsibly, demonstrating the value of sequencing for exploring complex sequence-function landscapes.
Date of Award27 Sept 2022
Original languageEnglish
Awarding Institution
  • University of Bristol
SupervisorTom Williams (Supervisor) & Thomas E Gorochowski (Supervisor)


  • Synthetic Biology
  • Genetic
  • Genetic circuit engineering
  • Sequencing
  • RNA
  • Bioinformatics
  • Responsible research and innovation
  • Conviviality
  • Microbiology
  • DNA

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