Investigating the Efficacy of Nanopore-Addressable Protein Tags as Reporters in Genetic Circuits

Student thesis: Master's ThesisMaster of Science by Research (MScR)

Abstract

Over the past few decades, huge leaps have been made in the complexity of engineered genetic circuits, as well as the ease with which they can be assembled. However, for them to become truly scalable, a new method of debugging is required that is less time-consuming and more cost-effective. The creation of nanopore-addressable protein tags (NTERs), genetic reporters that can be detected on a nanopore sequencer such as MinION, could potentially bring us closer to this possibility. However, until now, these protein tags had never been incorporated into a genetic circuit. In this thesis, I investigate the effect of incorporating NTERs into a genetic circuit. By first assembling two basic NOT-gate circuits, and then performing plate reader assays on cells containing those circuits, I am able to quantify the effect these NTERs had on circuit functionality. Fluorescence data gathered from the plate reader assays shows an increase in fluorescence intensity as the concentration of IPTG increases, which is the opposite of what was expected given the repression mechanisms of the circuit. This indicates a potential issue with either the inducer stock or the assembly of the circuit itself. In addition to this, the data shows an almost 10-fold increase in fluorescence intensity in the control circuits when compared to that of the NTER-containing NOT-gate circuits. With the only difference between these circuit being the presence of an NTER coding sequence, this stark difference in fluorescence intensity may be an indication of potential negative interactions between the NTER and the repressor to which it is fused. Despite my results being inconclusive, they have helped to clearly define which parts of the experimental procedure are most likely to cause issues when it comes to data collection, namely the assembly of the experimental circuits and the induction of gene expression during the plate reader assays. Any future attempts at establishing the efficacy of NTERs as genetic reporters should first focus on finding solutions for these problem areas.
Date of Award19 Mar 2024
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorThomas E Gorochowski (Supervisor) & Gary L A Barker (Supervisor)

Keywords

  • NTER
  • synthetic biology
  • Genetic circuit engineering
  • reporter protein
  • nanopore sequencing

Cite this

'