Degree Type

Dissertation

Date of Award

2021

Degree Name

Doctor of Philosophy

Department

Chemical and Biological Engineering

Major

Chemical Engineering

First Advisor

Thomas J. Mansell

Abstract

This dissertation contains six chapters that will focus on the effort of engineering E. coli Nissle 1917 (EcN) as a chassis that can be utilized by researchers to develop a new strain of probiotic drugs for treatments against gut-related diseases. Chapter 2 of this dissertation will discuss the components of synthetic biology that have been used in the past and can be used in the future to facilitate the process of engineering new strain of EcN as live biotherapeutics. In addition to that, the chapter will review current and past attempts in engineering EcN as probiotic drugs for treatments against diseases such as pathogenic infections, inherited metabolic disorders, and intestinal bowel diseases. Unfortunately, not many of these attempts are suitable to enter human clinical trials nor eligible to be used as a treatment for targeted illnesses. These are due to problems such as low level of protein expression, presence of antibiotic resistant genes, not addressing bio-containment guidelines, and lack of tools or platform to analyze the performance of engineered bacteria. Therefore, chapter 4, chapter 5, and chapter 6 of this dissertation will address some of the issues discussed in chapter 2.

Chapter 3 will review past, present, and future tools and technologies used to engineer bacteria at a genome-scale level. A genome-scale study is a process where collections of genes (genotypes) are mapped to certain traits (phenotypes) by implementing mutation to genes in the genome to study traits related to either loss-of-function or gain-of-function information. This knowledge can be used to engineer a superior mutant at genome-scale with improved phenotypes to carry out a specific task. High throughput technologies are the highlight of this chapter as it enables more mutants to be developed at a faster rate with high accuracy. The chapter will also discuss limitations in some of the tools used in genome-scale studies and the growing efforts to address them.

EcN has two naturally occurring cryptic plasmids that contain genes neither related to its probiotics traits nor associated with its fitness and survival attributes. If these plasmids are removed from EcN, it might alleviate the burden of having additional foreign genes in EcN without compromising the probiotic properties of this organism. Thus, the new strain will be able to produce a higher level of protein expression as compared to the wild type. Chapter 4 of this dissertation will demonstrate the curing process of these cryptic plasmids. Unfortunately, there were no differences observed in protein expression between plasmid cured EcN and wild type EcN (plasmids intact) when a high strength promoter was being used to express the florescent protein. However, after the publication of chapter 4, plasmid cured EcN was observed to produce a significantly higher protein expression as compared to wildtype EcN when a low-strength promoter was being used to express the gene of interest. This supports the initial hypothesis of this study. These two conflicted results occur because a high strength promoter can cause mRNA transcripts to reach saturation in this organism, hence causing both strains to have a similar level of protein expression. This means plasmid cured EcN might serve as a good strain to be used for expressing foreign genes under an uncharacterized low strength promoter. Apart from that, the curing plasmid process that was demonstrated in chapter 4 can serve as useful guidelines for anyone who wished to engineer plasmid-cured bacteria.

There are two methods of expressing genes of interest in bacteria; 1) cloning, which uses DNA plasmids to carry foreign genes, or 2) recombineering, which introduces foreign genes in the bacterial genome. Both methods use antibiotic-resistant genes as selection markers to screen for the correct insertion. In recombineering, the antibiotic resistant gene can be removed using frt site and FLP recombinase, but cloning using plasmid DNA requires the usage of an antibiotic as a selective pressure for the bacteria to maintain the foreign DNA. However, antibiotic-resistant genes can be transferred to other bacteria via horizontal gene transfer and this contributes to the emergence of MDR pathogen, but, most researchers still prefer to use plasmid DNA for introducing therapeutic genes into bacteria because it can provide a high-level protein expression as plasmids offer a higher copy number (10-500) as compared to expressing genes in the genome, which usually has a maximum of two copies in a bacteria. In chapter 5, this issue is addressed by utilizing two cryptic plasmids in EcN for protein expression. In chapter 4, the cloned cryptic plasmid, pMUT2-bla was found to be stable in EcN for two weeks when sub-cultured daily without antibiotic. Hence, we utilized the frt site and FLP recombinase to remove the antibiotic resistant gene in the cloned cryptic plasmid. A flow process for expressing protein of interest using the cryptic plasmids in EcN without utilizing antibiotic and antibiotic resistant gene is outlined in this chapter, serving as guidelines for researchers who wish to generate a new strain of EcN as probiotic drugs. In addition to that, we have engineered new EcNT7 strains that can use the PT7 promoter for a maximum level of protein expression. Strain EcNcBT7v31 is shown to provide a high expression level of bacteriocin, Microcin L, similar to E. coli BL21(DE3), which is an industrial standard for high-level protein expression, in agar diffusion assays. In this chapter, both the EcNcT7 (plasmid cured EcN with T7RNAP gene) strains and the cryptic plasmid system (pMUT2) were combined to establish the pMUT2-EcNcT7 platform that allows researchers to generate a new probiotic strain that is capable of expressing a high-level of therapeutic protein without using antibiotics and antibiotic-resistant genes.

Laboratory testing is extremely crucial for analyzing the safety and performance of engineered EcN. Preliminary testing of bacteria is usually performed in vitro using assays on test tubes or plates. Once the results of in vivo trials are confirmed positive, testing must be performed in vivo using animal models before entering the human clinical trials. In vivo testing has always been a gold standard in measuring the safety and performance of bacteria, however, animal models are not good enough for studies that require a close-up real-time observation, especially in studies related to gut-microbial interaction. In chapter 6, my colleague and I have designed an ex vivo platform that aims to provide researchers close monitoring of bacteria within a laboratory-cultured intestine, known as an organoid. An organoid is a mini small-intestine grown from stem cells harvested from mice intestine that is cultured in laboratory settings. Organoids can be re-grown in few passages for ex-vivo testing, thus this means the model can reduce the number of animals used for a particular study. In this project, we utilized the polymeric Ig receptor (pIgR)-mediated transcytosis pathway to deliver EcN into the luminal side of the organoid. The pIgR pathway functions to deliver polymeric immunoglobulin such as dIgA and pIgM from the basal membrane to the luminal side of the intestinal. We have coated the outer layer of EcN with mice serum containing pIgM in order to deliver the bacteria into the luminal side of organoid. Data shown chapter 6 have verify that there is a possibility that the serum-coated EcN is transcytosis from the basal side to the luminal side of the organoid, which allows EcN to be cultured within the organoid.

DOI

https://doi.org/10.31274/etd-20210609-205

Copyright Owner

Halimatun Sakdiah binti Zainuddin

Language

en

File Format

application/pdf

File Size

169 pages

Available for download on Tuesday, June 07, 2022

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