Continuous Measurement of Biological Noise in Escherichia Coli Using Time-lapse Microscopy

Summary

This work presents a microscopy method that allows live imaging of a single cell of Escherichia coli for analysis and quantification of the stochastic behavior of synthetic gene circuits.

Abstract

The protocol developed here offers a tool to enable computer tracking of Escherichia coli division and fluorescent levels over several hours. The process starts by screening for colonies that survive on minimal media, assuming that only Escherichia coli harboring the correct plasmid will be able to thrive in the specific conditions. Since the process of building large genetic circuits, requiring the assembly of many DNA parts, is challenging, circuit components are often distributed between multiple plasmids at different copy numbers requiring the use of several antibiotics. Mutations in the plasmid can destroy transcription of the antibiotic resistance genes and interject with resources management in the cell leading to necrosis. The selected colony is set on a glass-bottom Petri dish and a few focus planes are selected for microscopy tracking in both bright field and fluorescent domains. The protocol maintains the image focus for more than 12 hours under initial conditions that cannot be regulated, creating a few difficulties. For example, dead cells start to accumulate in the lenses' field of focus after a few hours of imaging, which causes toxins to buildup and the signal to blur and decay. Depletion of nutrients introduces new metabolic processes and hinder the desired response of the circuit. The experiment's temperature lowers the effectivity of inducers and antibiotics, which can further damage the reliability of the signal. The minimal media gel shrinks and dries, and as a result the optical focus changes over time. We developed this method to overcome these challenges in Escherichia coli, similar to previous works developing analogous methods for other micro-organisms. In addition, this method offers an algorithm to quantify the total stochastic noise in unaltered and altered cells, finding that the results are consistent with flow analyzer predictions as shown by a similar coefficient of variation (CV).

Introduction

Synthetic biology is a multidisciplinary field that has emerged in the past decade and aims to translate engineering design principles into rational biological design^1,2,3, in an effort to achieve multi-signal integration and processing in living cells for understanding the basic science^4,5, diagnostic, therapeutic and biotechnological applications^6,7,8,9,10

Protocol

1. Media and culture preparation

1. Prepare stock solution of 1,000x Carbenicillin (50 mg/mL) or relevant antibiotic.
   1. .Weigh 0.5 g of carbenicillin. Add 10 mL of sterile H₂O. Dissolve completely.
   2. Sterilize carbenicillin stock through a 0.22 µm syringe filter. Aliquot the antibiotic solution and store at -20 °C.
2. To prepare lysogeny broth (LB) plates, mix 5 g of tryptone, 5 g of NaCl, 2.5 g of yeast extract and 7.5 g of .......

Discussion

In this work, we developed a protocol that enables computer tracing of Escherichia coli live cells, following division and fluorescent levels over a period of hours. This protocol allows us to quantify the stochastic dynamics of genetic circuits in Escherichia coli by measuring the CV and SNR in real time. In this protocol, we compared the stochastic behaviors of two different circuits as shown in Figure 10. It has been shown that plasmids with low copy numbers are more prone to stochas.......

Materials

Name	Company	Catalog Number	Comments
35mm glass dish	mattek	P35G-0.170-14-C	thickness corresponding with microscope lense.
Agarose	Lonza	5004	LB preperation
AHL	Sigma-Aldrich	K3007	inducer
Bacto tryptone	BD - Becton, Dickinson and Company	211705	LB preperation
Carb	Invitrogen	10177-012	antibiotic
Carb	Formedium	CAR0025	antibiotic
Casamino acids	BD - Becton, Dickinson and Company	223050	minimal media solution
eclipse Ti	nikon		inverted microscope
Glucose	Sigma-Aldrich	G5767	minimal media solution
Glyserol	Bio-Lab	000712050100	minimal media substrate
Immersol 518F	zeiss	4449600000000	immersion oil
M9 salt solution	Sigma-Aldrich	M6030	minimal media solution
NaCl	Bio-Lab	214010	LB preperation
Noble agar	Sigma-Aldrich	A5431	minimal media substrate
parafilm tape	Bemis	PM-996	refered to as tape in text
Seaplaque GTG Agarose	Lonza	50111	minimal media substrate
thaymine B1	Sigma-Aldrich	T0376	minimal media solution
Yeast Extract	BD - Becton, Dickinson and Company	212750	LB preperation