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Serine Integrase-Based Event Recording in E. coli


Shur, Andrey Sergeyvich (2022) Serine Integrase-Based Event Recording in E. coli. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/x4q0-nx18.


DNA is a unique molecule that has evolved to serve as the genetic material for life. It seems straightforward to consider this molecule not only as a wonder of the natural world but as a tool for information storage and retrieval. Bacteria have evolved to conserve DNA, but bacteriophages have evolved to specifically integrate their genomes using integrases. In response to viruses, bacteria have evolved the RNA-guided nuclease Cas9 to destroy viral DNA before it can be integrated. The fruits of these evolutionary pressures prove useful to the researcher interested in easily editing DNA. In this work, we have engineered a genetic circuit that can enact specific and controlled genetic changes in response to changing small molecule concentrations. Known DNA sequences can be repeatedly integrated into a synthetic array such that their identity and order encodes information about past small molecule concentrations that the cell has experienced. To accomplish this, we use catalytically inactive CRISPR-Cas9 (dCas9) to bind to and block attachment sites for the integrase Bxb1. Through the co-expression of dCas9 and guide RNA, Bxb1 can be directed to integrate one of two engineered "ink" plasmids, which correspond to two orthogonal small molecule inducers whose presence or absence as a function of time can be recorded with this system. Integrase sites present on these plasmids are found to not participate in intramolecular "deletion" reactions if closer than 100 bp. Guide RNAs overlapping integrase attachment sites are found to effectively block integrase activity at those sites if the overlap is equal to 9 or 19 base pairs. Other overlap values, including forward or reverse binding result in ineffective integrase activity repression. We develop 8 orthogonal guide RNA sequences capable of binding to and repressing integrase activity at the attP site. Plasmid multimers are sequenced using Oxford Nanopore sequencing and found to follow population-level predictions of event record identity. Single DNA states are found insufficient for identifying past history of events; an ensemble of DNA states at the population level must be used. A modular modeling framework is developed (Global enumeration) to describe this system, and integrated with the existing chemical reaction network creation automation software BioCRNpyler. The modeling framework developed here automatically creates chemical reaction networks based on typical linear DNA-based synthetic biology "genetic constructs" and predicts transcripts and proteins produced based on simple transcription/translation rules. Integrase-based recombination events can also be predicted in a recursive way.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:synthetic biology; event recording; genetic memory; molecular biology; biochemistry; integrases; recombinases; long-read sequencing;
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Bioengineering
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Murray, Richard M.
Thesis Committee:
  • Elowitz, Michael B. (chair)
  • Pierce, Niles A.
  • Sternberg, Paul W.
  • Murray, Richard M.
Defense Date:13 December 2021
Non-Caltech Author Email:andrey.shur (AT)
Record Number:CaltechTHESIS:12212021-193826426
Persistent URL:
Related URLs:
URLURL TypeDescription ItemSoftware tools used/developed in this work ItemSoftware tools used/developed in this work integrated into chapter 2 integrated into chapter 2 3 is used in supplemental material of this article
Shur, Andrey Sergeyvich0000-0001-9372-6713
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:14455
Deposited By: Andrey Shur
Deposited On:20 Jan 2022 21:00
Last Modified:13 Jul 2022 18:54

Thesis Files

[img] PDF (thesis) - Final Version
See Usage Policy.

[img] MS Excel (List of construct sequences used in the thesis) - Supplemental Material
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