Computational Methods in Synthetic Biology Mario

Andrea Marchisio pdf download

tps://textbookfull.com/product/computational-methods-in-synthetic-biology-mario-andrea-marchisio/

★★★★★ 4.6/5.0 (31 reviews) ✓ 186 downloads ■ TOP RATED

"Perfect download, no issues at all. Highly recommend!" - Mike D.

DOWNLOAD EBOOK
 Computational Methods in Synthetic Biology Mario Andrea
Marchisio pdf download

TEXTBOOK EBOOK TEXTBOOK FULL

Available Formats

■ PDF eBook Study Guide TextBook

EXCLUSIVE 2025 EDUCATIONAL COLLECTION - LIMITED TIME

INSTANT DOWNLOAD VIEW LIBRARY

Collection Highlights

Computational Methods in Synthetic Biology 2nd Edition

Mario Andrea Marchisio

Synthetic Biology Methods and Protocols Methods in

Molecular Biology 2760 2nd Edition Braman Jeffrey Carl Edt

Epithelial Cell Culture Methods and Protocols Methods in

Molecular Biology 2749 Mario Baratta

Synthetic Biology Methods and Protocols 2nd Edition

Jeffrey Carl Braman
Mining Lurkers in Online Social Networks Principles Models
and Computational Methods Andrea Tagarelli

Systems Biology Application in Synthetic Biology 1st

Edition Shailza Singh (Eds.)

Synthetic Biology Jeffrey Carl Braman

Computational Cell Biology Methods and Protocols Louise

Von Stechow

Computational Stem Cell Biology Methods and Protocols

Patrick Cahan
Methods in
Molecular Biology 2189

Mario Andrea Marchisio Editor

Computational
Methods in
Synthetic Biology
Second Edition
METHODS IN MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK

For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
with a helpful notes section offering tips and tricks of the trade as well as troubleshooting
advice. These hallmark features were introduced by series editor Dr. John Walker and
constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
indexed in PubMed.
 Computational Methods
in Synthetic Biology
Second Edition

Edited by

Mario Andrea Marchisio

School of Pharmaceutical Science and Technology, Tianjin University, Tianjin, China
Editor
Mario Andrea Marchisio
School of Pharmaceutical Science
and Technology
Tianjin University
Tianjin, China

ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-0821-0 ISBN 978-1-0716-0822-7 (eBook)
https://doi.org/10.1007/978-1-0716-0822-7

© Springer Science+Business Media, LLC, part of Springer Nature 2015, 2021

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply,
even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations
and therefore free for general use.
The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been
made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer
Nature.
The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A.
Preface

The second edition of the book Computational Methods in Synthetic Biology can be seen as
complementary to the first edition. Almost all chapter authors are new, and the overlap
between the topics covered in the two editions is also relatively small.
The continuity with the first edition is, mainly, in the first part of the book that contains
chapters on foundational themes in Synthetic Biology such as biological parts and gene
circuits design.
As a novelty with respect to the first edition, this book hosts a part dedicated to
computational tools for the design and analysis of CRISPR-Cas systems, now largely
adopted in synthetic biology. Another part completely new with respect to the first edition
is on “synthetic genomics”: it presents two chapters, one about the assembly of synthetic
chromosomes (great progress has been made, in recent years, in the construction of artificial
S. cerevisiae chromosomes) and the other about minimal genome design (another funda-
mental goal of synthetic biology). Moreover, in this second edition, more emphasis is given
to methods for the analysis of metabolic pathways and techniques borrowed from Systems
Biology and Electrical Engineering to study and optimize various kinds of synthetic
networks.
We think that, on the whole, the two editions of our book give a very broad overview of
the research areas that can be met in the area of in silico synthetic biology.

Tianjin, China Mario Andrea Marchisio

v
Contents

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Using a Design of Experiments Approach to Inform the Design

of Hybrid Synthetic Yeast Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
James Gilman, Valentin Zulkower, and Filippo Menolascina
2 Computational Design of Multiplex Oligonucleotide-Based Assays. . . . . . . . . . . . 19
Michaela Hendling and Ivan Barišić
3 Computational Methods for the Design of Recombinase Logic Circuits . . . . . . . 31
Sarah Guiziou and Jerome Bonnet
4 Modular Modeling of Genetic Circuits in SBML Level 3 . . . . . . . . . . . . . . . . . . . . 45
Mario Andrea Marchisio
5 CRISPR-ERA: A Webserver for Guide RNA Design of Gene
Editing and Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Honglei Liu and Xiaowo Wang
6 iGUIDE Method for CRISPR Off-Target Detection . . . . . . . . . . . . . . . . . . . . . . . . 71
Christopher L. Nobles
7 Web-Based Base Editing Toolkits: BE-Designer and BE-Analyzer . . . . . . . . . . . . . 81
Gue-Ho Hwang and Sangsu Bae
8 Synthetic Gene Circuit Analysis and Optimization . . . . . . . . . . . . . . . . . . . . . . . . . . 89
Irene Otero-Muras and Julio R. Banga
9 Monitoring Single S. cerevisiae Cells with Multifrequency
Electrical Impedance Spectroscopy in an Electrode-Integrated
Microfluidic Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
Zhen Zhu, Yangye Geng, and Yingying Wang
10 Construction of Protein Expression Network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Nor Afiqah-Aleng and Zeti-Azura Mohamed-Hussein
11 Systems-Theoretic Approaches to Design Biological Networks
with Desired Functionalities. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Priyan Bhattacharya, Karthik Raman, and Arun K. Tangirala
12 A Deterministic Compartmental Modeling Framework for Disease
Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
King James B. Villasin, Eva M. Rodriguez, and Angelyn R. Lao
13 Computer Aided Assembly and Verification of Synthetic
Chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Giovanni Stracquadanio and Valentin Zulkower
14 Minimal Genome Design Algorithms Using Whole-Cell Models . . . . . . . . . . . . . 183
Joshua Rees-Garbutt, Oliver Chalkley, Claire Grierson,
and Lucia Marucci

vii
viii Contents

15 Tn-Core: Functionally Interpreting Transposon-Sequencing

Data with Metabolic Network Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
George C. diCenzo, Marco Galardini, and Marco Fondi
16 Genome-Scale Metabolic Modeling of Escherichia coli and Its
Chassis Design for Synthetic Biology Applications . . . . . . . . . . . . . . . . . . . . . . . . . . 217
Bashir Sajo Mienda and Andreas Dr€ ager
17 A Systems Bioinformatics Approach to Interconnect Biological Pathways . . . . . . 231
George Minadakis and George M. Spyrou

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Contributors

NOR AFIQAH-ALENG • Institute of Systems Biology (INBIOSIS), Universiti Kebangsaan

Malaysia, Bangi, Selangor, Malaysia; Institute of Marine Biotechnology, Universiti
Malaysia Terengganu, Kuala Nerus, Terengganu, Malaysia
SANGSU BAE • Department of Chemistry, Hanyang University, Seoul, South Korea; Research
Institute for Convergence of Basic Sciences, Hanyang University, Seoul, South Korea
JULIO R. BANGA • BioProcess Engineering Group, IIM-CSIC, Spanish National Research
Council, Vigo, Spain
IVAN BARIŠIĆ • Center for Health & Bioresources, Molecular Diagnostics, Austrian Institute
of Technology GmbH, Vienna, Austria
PRIYAN BHATTACHARYA • Department of Chemical Engineering, Indian Institute of
Technology Madras, Chennai, India
JEROME BONNET • Centre de Biochimie Structurale (CBS), INSERM U1054, CNRS
UMR5048, University of Montpellier, Montpellier, France
OLIVER CHALKLEY • Department of Engineering Mathematics, University of Bristol, Bristol,
UK; Bristol Centre for Complexity Science, University of Bristol, Bristol, UK
GEORGE C. DICENZO • Department of Biology, Queen’s University, Kingston, ON, Canada
ANDREAS DRAGER€ • Computational Systems Biology of Infection and Antimicrobial-
Resistant Pathogens, Institute for Bioinformatics and Medical Informatics (IBMI),
University of Tübingen, Tübingen, Germany; Department of Computer Science, University
of Tübingen, Tübingen, Germany; German Center for Infection Research (DZIF), partner
site Tübingen, Tübingen, Germany
MARCO FONDI • Department of Biology, University of Florence, Sesto Fiorentino, FI, Italy
MARCO GALARDINI • Biological Design Center, Boston University, Boston, MA, USA;
Department of Biomedical Engineering, Boston University, Boston, MA, USA
YANGYE GENG • Key Laboratory of MEMS of Ministry of Education, Southeast University,
Nanjing, China
JAMES GILMAN • Institute for Bioengineering, School of Engineering, University of
Edinburgh, Edinburgh, UK
CLAIRE GRIERSON • BrisSynBio, University of Bristol, Bristol, UK; School of Biological
Sciences, University of Bristol, Bristol, UK
SARAH GUIZIOU • Centre de Biochimie Structurale (CBS), INSERM U1054, CNRS
UMR5048, University of Montpellier, Montpellier, France; Department of Biology,
University of Washington, Seattle, WA, USA
MICHAELA HENDLING • Center for Health & Bioresources, Molecular Diagnostics, Austrian
Institute of Technology GmbH, Vienna, Austria
GUE-HO HWANG • Department of Chemistry, Hanyang University, Seoul, South Korea;
Research Institute for Convergence of Basic Sciences, Hanyang University, Seoul, South
Korea
ANGELYN R. LAO • Mathematics and Statistics Department, De La Salle University, Manila,
Philippines
HONGLEI LIU • School of Biomedical Engineering, Capital Medical University, Beijing,
China; Beijing Key Laboratory of Fundamental Research on Biomechanics in Clinical
Application, Capital Medical University, Beijing, China

ix
x Contributors

MARIO ANDREA MARCHISIO • School of Pharmaceutical Science and Technology, Tianjin

University, Tianjin, People’s Republic of China
LUCIA MARUCCI • BrisSynBio, University of Bristol, Bristol, UK; Department of Engineering
Mathematics, University of Bristol, Bristol, UK; School of Cellular and Molecular Medicine,
University of Bristol, Bristol, UK
FILIPPO MENOLASCINA • Institute for Bioengineering, School of Engineering, University of
Edinburgh, Edinburgh, UK
BASHIR SAJO MIENDA • Department of Microbiology & Biotechnology, Faculty of Science,
Federal University Dutse, Dutse, Jigawa, Nigeria
GEORGE MINADAKIS • Department of Bioinformatics, The Cyprus School of Molecular
Medicine, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus
ZETI-AZURA MOHAMED-HUSSEIN • Institute of Systems Biology (INBIOSIS), Universiti
Kebangsaan Malaysia, Bangi, Selangor, Malaysia; Department of Applied Physics, Faculty
of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia
CHRISTOPHER L. NOBLES • Department of Microbiology, Perelman School of Medicine,
University of Pennsylvania, Philadelphia, PA, USA
IRENE OTERO-MURAS • BioProcess Engineering Group, IIM-CSIC, Spanish National
Research Council, Vigo, Spain
KARTHIK RAMAN • Department of Biotechnology, Bhupat and Jyoti Mehta School of
Biosciences, Indian Institute of Technology Madras, Chennai, India; Initiative for
Biological Systems Engineering, Indian Institute of Technology Madras, Chennai, India;
Robert Bosch Centre for Data Science and Artificial Intelligence (RBC-DSAI), Indian
Institute of Technology Madras, Chennai, India
JOSHUA REES-GARBUTT • BrisSynBio, University of Bristol, Bristol, UK; School of Biological
Sciences, University of Bristol, Bristol, UK
EVA M. RODRIGUEZ • Department of Mathematics, School of Sciences and Engineering,
University of Asia and the Pacific, Pasig City, Philippines
GEORGE M. SPYROU • Department of Bioinformatics, The Cyprus School of Molecular
Medicine, The Cyprus Institute of Neurology & Genetics, Nicosia, Cyprus
GIOVANNI STRACQUADANIO • School of Biological Sciences, The University of Edinburgh,
Edinburgh, UK
ARUN K. TANGIRALA • Department of Chemical Engineering, Indian Institute of Technology
Madras, Chennai, India; Robert Bosch Centre for Data Science and Artificial Intelligence
(RBC-DSAI), Indian Institute of Technology Madras, Chennai, India
KING JAMES B. VILLASIN • Department of Mathematics, School of Sciences and Engineering,
University of Asia and the Pacific, Pasig City, Philippines
XIAOWO WANG • Ministry of Education Key Laboratory of Bioinformatics, Tsinghua
University, Beijing, China; Center for Synthetic and Systems Biology, Tsinghua University,
Beijing, China; Bioinformatics Division, Beijing National Research Center for
Information Science and Technology, Tsinghua University, Beijing, China; Department of
Automation, Tsinghua University, Beijing, China
YINGYING WANG • Key Laboratory of MEMS of Ministry of Education, Southeast University,
Nanjing, China
ZHEN ZHU • Key Laboratory of MEMS of Ministry of Education, Southeast University,
Nanjing, China
VALENTIN ZULKOWER • Edinburgh Genome Foundry, The University of Edinburgh,
Edinburgh, UK
 Chapter 1

Using a Design of Experiments Approach to Inform

the Design of Hybrid Synthetic Yeast Promoters
James Gilman, Valentin Zulkower, and Filippo Menolascina

Abstract
Hybrid promoter engineering takes advantage of the modular nature of eukaryotic promoters by combin-
ing discrete promoter motifs to confer novel regulatory function. By combinatorially screening sequence
libraries for trans-acting transcriptional operators, activators, repressors and core promoter sequences, it is
possible to derive constitutive or inducible promoter collections covering a broad range of expression
strengths. However, combinatorial approaches to promoter design can result in highly complex, multidi-
mensional design spaces, which can be experimentally costly to thoroughly explore in vivo. Here, we
describe an in silico pipeline for the design of hybrid promoter libraries that employs a Design of Experi-
ments (DoE) approach to reduce experimental burden and efficiently explore the promoter fitness land-
scape. We also describe a software pipeline to ensure that the designed promoter sequences are compatible
with the YTK assembly standard.

Key words Design of Experiments, Hybrid promoter engineering, JMP, Synthetic promoter, Yeast

1 Introduction

Robust, predictable genetic parts are a fundamental requirement if

scalable, bottom-up engineering of complex biological systems is to
be achieved [1, 2]. Multiple decision variables are available to tune
synthetic gene circuits [3], but on a practical level the control of
transcription activation using well-characterized promoters with
defined activation and output functions (or “strengths”) offers a
simple, widely used method by which the expression of transgenes
or pathways can be balanced [1, 3].
Numerous approaches are available for the identification and
design of promoters with desirable characteristics. For example,
endogenous promoter sequences may be identified using genomic
or transcriptomic analyses of the host organism of interest, fol-
lowed by extensive in vivo characterization of promoter activity in
multiple genetic and environmental contexts [3, 4]. However, nat-
ural promoter activity is often highly context specific [5] and

Mario Andrea Marchisio (ed.), Computational Methods in Synthetic Biology, Methods in Molecular Biology, vol. 2189,
https://doi.org/10.1007/978-1-0716-0822-7_1, © Springer Science+Business Media, LLC, part of Springer Nature 2021

1
2 James Gilman et al.

subject to interaction with a multitude of endogenous regulatory

systems [6], complicating prediction of activity levels under varying
conditions. Furthermore, the repeated use of identical promoter
sequences is complicated in eukaryotes as a result of the efficient
homologous recombination systems that are present in many spe-
cies, which can decrease the stability of recombinant DNA con-
structs [7, 8].
Given the inherent limitations of endogenous promoters, the
rational design of synthetic sequences with defined transcriptional
activities has become a widely used method for the development of
synthetic biology parts [3, 5, 9]. Although the regulation of
eukaryotic transcription is a complex process, involving DNA–pro-
tein interactions between promoters and highly specific transcrip-
tion factors, enhancers, and suppressors [10], promoter elements
themselves can be abstracted to somewhat modular combinations
of key motifs [9] that provide binding sites for trans-acting regu-
latory proteins [8]. As a result of this inherent modularity, eukary-
otic promoters are particularly suited to hybrid engineering
approaches, in which well-characterized promoter sequence motifs
are combined to confer novel functionality [11–15]. For example, a
2018 study by Dossani et al. used a combinatorial approach to
design a library of hybrid promoters that are regulated by a syn-
thetic transcription factor [16]. The authors used a full-factorial
approach to hybrid promoter design, in which all possible combi-
nations of the sequence motifs of interest were characterized
(Fig. 1). Short (100 bp) or long (250 bp) variants of 10 core
promoters were combined with either 1, 2, or 3 tandem repeats
of 1 of 4 operator sequences of the LexA DNA binding domain. A
library of 240 putative synthetic promoters was therefore designed
in silico, of which 154 sequences were successfully characterized
in vivo [16].
A full-factorial approach to hybrid promoter design rapidly
becomes an inefficient, experimentally expensive way to explore
the promoter fitness landscape as the complexity of the promoter
structure increases. To restrict the complexity of their design space,
Dossani et al. specified that only one type of operator could be
present in any single promoter sequence [16]. However, such a
restriction prevents exploration of regions of the design space that
may encode promoters with desirable characteristics [15, 17], and
would not be possible when designing promoters that respond to
multiple input signals, which by their nature require multiple
operators [18, 19]. For a hypothetical hybrid promoter containing
x possible motifs, each with y possible variants, yx experiments
would be required to satisfy a full-factorial design [20].
In lieu of designing full-factorial hybrid promoter libraries, a
multifactorial Design of Experiments (DoE) approach to promoter
design offers a method by which a rationally selected subset of
putative promoter sequences can be identified in silico to maximize
 Design of Experiments for Synthetic Promoter Design 3

Operator Core promoter

1x Short (100 bp)

CUP1p LEU2p
2x Consensus colE1 Long (250 bp)
3x
GAL1p SPO13p

umuDC uvrA
GCN4p SSL1p

HEM13p TEF1p

HHF2p ZRT1p

Fig. 1 Diagrammatic representation of the promoter structure characterized by Dossani et al.

a. b.
Factor 1

Factor 1
3

3
or

or
ct

ct
Fa

Fa

Factor 2 Factor 2

Fig. 2 Exploring a design space using (a) a full-factorial experimental design or (b) a Taguchi array. A Taguchi
array is a type of fractional factorial experimental design that is balanced, so that all levels of the experimental
factors of interest are evaluated equally [21]

the proportion of the design space that is explored whilst experi-

mental burden is minimized (Fig. 2) [22]. Subsequent in vivo
empirical characterization of the designed sequences can provide
well-structured data sets against which statistical models can be
trained [23], allowing the promoter fitness landscape to be mapped
[24] and promoter sequences to be optimized to provide defined
strengths. This design-build-test cycle can be further expedited
using BioFoundries to facilitate the high-throughput in vitro
4 James Gilman et al.

assembly of the designed constructs [25]. Here, we describe a

generally applicable protocol for the in silico design of a hybrid
promoter library using DoE methodologies and the preparation of
the resulting sequences for DNA synthesis and assembly on a
BioFoundry platform, using the Saccharomyces cerevisiae hybrid
promoter library described by Dossani et al. [16] as an example.

2 Materials

1. JMP Pro statistical analysis software (SAS Institute Inc., www.

jmp.com).
2. Joint Genome Institute build-optimization software tools
(BOOST) [26]; see Note 1.
3. Edinburgh Genome Foundry Collection of Useful Biological
Apps (CUBA; https://cuba.genomefoundry.org/).

3 Methods

An overview of the Design of Experiments approach to promoter

design, in both general terms and as applied to the promoter design
space identified by Dossani et al., is provided in Fig. 3.

3.1 Identifying Hybrid promoter engineering requires the careful selection of can-
Candidate Promoter didate sequence motifs so that the final promoter library contains
Motifs sequences with the desired functionality. However, the number and
type of motifs that are included in an experimental design will be
heavily dependent on the requirements of individual studies. In
particular, a library of constitutive promoters will comprise a differ-
ent selection of functional motifs to a library of inducible promoter
sequences. The promoter sequence motifs considered in the library
designed by Dossani et al. are summarized in Fig. 1.
Regardless if a hybrid promoter library is intended to be con-
stitutive or inducible, core promoter sequences are required to
determine the start site and direction of transcription [5], and to
provide a “backbone” to which upstream enhancers, operators, or
suppressors can be added. Synthetic minimal core promoters with a
range of strengths have been previously described and could be
applied in hybrid promoter design [27]. Alternatively, putative core
promoters can be bioinformatically identified from upstream of
well-characterized genes in the host organism of interest [16], or
previously characterized core promoters can be extracted from
synthetic biology part registries such as the iGEM Registry of
Standard Biological Parts [28] or SynBioHub [29]. The endoge-
nous function of candidate core promoter sequences should be
carefully considered, as this is likely to impact the activity of the
 Design of Experiments for Synthetic Promoter Design 5

Fig. 3 Flow chart outlining the Design of Experiments approach to hybrid

promoter design. The process is described (a) in general terms and (b) as
applied to the promoter design space identified by Dossani et al.
6 James Gilman et al.

hybrid promoter library. For example, when designing inducible

promoters, it may be desirable to select core promoters from
upstream of genes that are known to be natively differentially
expressed in the presence or absence of the inducer ligand or
environmental conditions of interest [16]. Alternatively, promoters
whose function remains constant across a wide range of environ-
mental or genetic contexts may be preferable [30]. In the case of
the hybrid promoter library designed by Dossani et al., the 10 core
promoter sequences that were chosen were isolated from upstream
of well-characterized genes from the S. cerevisiae genome, with the
stated aim of “representing both constitutive and inducible profiles
at both low and high expression levels” [16].
Upstream of the core promoter, enhancer, suppressor and
operator motifs can be specified to confer various functions. If,
for example, the aim of a study is to develop a library of promoters
that cause high levels of transcription activation, the hybrid pro-
moter design could include natural or synthetic Upstream Activa-
tion Sequences (UAS), as tandem repeats or combinations of UAS
can be used to tune the dynamic range of promoter activity [15].
To confer inducibility to a synthetic promoter, operator
sequences are required that facilitate DNA–protein interactions
between the promoter sequence and the relevant transcription
factor, and promoter function can be fine-tuned by combining
multiple operators or by modifying the operator sequence [31–
33]. Native transcription factor binding sites can be individually
identified from individual endogenous promoters with well-
characterized activity [11, 13], or isolated en masse from ChIP-
seq-derived data sets [33, 34]. As an alternative to co-opting
endogenous regulatory machinery and to reduce cross-talk
between native and synthetic genes or circuits, synthetic transcrip-
tion factors and operator sequences can be incorporated into hybrid
promoter design. Synthetic transcription factors can be targeted to
promoters using zinc fingers and their associated binding sequences
[31], Transcription Activator Like Effectors (TALEs) [30], catalyt-
ically inactive cas9 (dCas9) [32, 35], or, as in the study performed
by Dossani et al., using prokaryotic DNA binding domains like
LexA [16].

3.2 Defining Once identified, promoter motifs can be used to define the factors
Experimental and responses of an experimental design. In the case of the hybrid
Parameters promoter library designed by Dossani et al., the experimental fac-
tors of interest are the identity and length of the core promoter and
the identity and copy number of the operator sequence (Fig. 1,
Table 1).
Dossani et al., defined two responses of interest in their experi-
mental design. The “Inducibility” of a promoter was defined as the
fold-change in promoter induction from 0 to 100 nM Estradiol,
and the promoter “Response” was defined as the maximum level of
 Design of Experiments for Synthetic Promoter Design 7

Table 1
Experimental factors for the hybrid promoter design specified by Dossani et al.

Pro_core Pro_core_length Operator Operator_count

GAL1p Short consensus 1
LEU2p Long colE1 2
SPO13p uvrA 3
TEF1p umuDC
HHF2p
GCN4p
CUP1p
HEM13p
ZRT1p
SSL1p

induction at 10 nM Estradiol [16]. When considering which

responses to characterize for a designed inducible system, it may
also be beneficial to consider the “Leakiness” of a promoter in the
absence of inducer [36] as a potential experimental response, as
promoters with minimal levels of background activity are often
desirable.

3.3 Screening Prior to DNA synthesis, commercial vendors screen submitted

the Promoter Design sequences for the presence of features that are known to be predic-
Space for Synthesis tive of synthesis failure [26]. Violation of these synthesis constraints
Constraint Violations can preclude the synthesis of certain DNA sequences. The inclusion
and Incompatible of one or more of these hard-to-synthesize sequences in an experi-
Restriction Sites mental design could result in missing data when the hybrid pro-
moter library is characterized in vivo, reducing the amount of the
promoter design space that is empirically explored and potentially
reducing the predictive power of any downstream models. To
prevent the inclusion of promoter sequences that violate synthesis
constraints in an experimental design, the design space of interest
should be screened for any problematic sequences, which can be
excluded in silico from subsequent experimental designs. Addition-
ally, the promoter sequences should be screened for the presence of
any restriction sites that would render them incompatible with
whichever DNA assembly strategy is to be used, e.g., BsaI restric-
tion sites in the case of the method discussed in Subheading 3.5.
Any illegal restriction sites can be removed from promoter
sequences in silico using silent mutations, or the promoter can be
included on the list of sequences to be excluded from the final
experimental design.
8 James Gilman et al.

1. Using the Full-Factorial Design function of JMP software,

generate a full combinatorial promoter library from the identi-
fied sequence motifs. Once the design is generated in JMP, the
find and replace function of any text editing software can be
used to convert the categorical motif labels into DNA sequence
to generate the complete promoter sequences.
2. Identify and remove any incompatible restriction sites from the
promoter sequences using the CUBA“Sculpt a Sequence” tool
(see Note 2). For example, MoClo-based assembly strategies
require that any candidate DNA parts do not contain BsaI
restriction sites.
3. Analyze the designed promoters using the “Polisher” function
of BOOST to identify sequences that violate DNA synthesis
constraints of the chosen synthesis vendor (see Note 3).
Dossani et al. identified 60 putative promoters that violated
the synthesis constraints specified by IDT and that were there-
fore likely to be problematic to synthesize.
4. Once screening is complete, JMP scripting language (JSL)
should be used to write a Disallowed Combinations script to
prevent the inclusion of any problematic sequences in down-
stream experimental designs. The script should take the form:

Pro_core = 5 & Pro_core_length = 2 & Operator = 2 & Operator_count

= 1 |

Pro_core = 4 & Pro_core_length = 2 & Operator = 2 & Operator_count

= 1 |

Pro_core = 7 & Pro_core_length = 2 & Operator = 1 & Operator_count

= 1 |

Pro_core = 7 & Pro_core_length = 1 & Operator = 1 & Operator_count

= 1

where the numbers refer to the ordinal value of the level

being excluded (Table 1). For example, the first line of the
above script refers to a promoter consisting of the HHF2p
core promoter, in its short form, combined with a single copy
of the colE1 operator sequence.
 Design of Experiments for Synthetic Promoter Design 9

3.4 Designing The Custom Design platform of JMP Pro can be used to design a
a Hybrid Promoter hybrid promoter library according to the following protocol. The
Library Custom Design window, populated with settings to generate an
experimental design based on the promoter structure described by
Dossani et al., [16] is shown in Fig. 4.
1. Specify the responses to be used in the experimental design,
and whether the goal of the experiment is to maximize or
minimize the value of each response. It is also possible to aim
to match a specified target value, or to have no goal for a
response if the purpose of the experiment is to explore the
design space of interest rather than to optimize promoter
performance.
2. Specify the factors to be used in the experimental design. The
factor type must also be specified; the majority of the factors
included in hybrid promoter design are likely to be categorical.
In the case of the hybrid promoter library designed by Dossani
et al., Pro_Core, Pro_core_length, and Operator were set as
categorical factors. Operator_count was defined as a Discrete
Numeric Factor. It is also possible to specify the difficulty of
changing a factor. Setting a factor as “Hard” to change will
impose blocking on the final experimental design. However, as
the promoter sequences designed by Dossani et al. were
synthesized, there were no constraints on changing factors
between runs. As such, all four experimental factors were set
as “Easy” to change. Once the experimental responses and
factors have been defined they can be saved as data tables.
This option can be found in the “Red Arrow” menu, which is
located at the top left of the Custom Design window (Fig. 4).
The resulting tables can subsequently be reloaded in any of the
JMP DoE platforms, expediting the process of generating
multiple experimental designs from the same responses and
factors.
3. Define the factor constraints to be applied to the experimental
design by inserting the Disallowed Combinations Script (see
Subheading 3.3).
4. Specify the effects that are to be estimated in the assumed
model (i.e., the model that will be trained on the experimental
data). Main effects are added by default, as are polynomial
terms if a Discrete Numeric factor has been specified. Interac-
tion effects up to the fifth order can be specified, as can Poly-
nomial terms for Continuous or Discrete Numeric factors,
again up to the fifth order. The Cross option can be used to
add specific interaction terms. As one of the aims of a Design of
Experiments approach to hybrid promoter design is a reduc-
tion in experimental burden as compared to a full-factorial
approach, a trade-off is required between the complexity of
10 James Gilman et al.

Fig. 4 JMP Pro version 14 DoE Custom Design window

 Design of Experiments for Synthetic Promoter Design 11

the specified model and the number of runs that can be experi-
mentally budgeted. A more complex model can potentially
yield greater insights into the system under investigation, but
generally requires a greater number of experimental runs to
resolve than a more parsimonious alternative. To prevent
designs becoming too experimentally costly, the Estimability
of model terms can be changed. The Estimability of a term is a
designation of the importance of estimating that term in the
final design. Setting the Estimability of a term as “Necessary”
will force the JMP algorithm to generate an experimental
design in which that term can be estimated, whereas setting
Estimability to “If Possible” will result in a design that esti-
mates the model term only if there is sufficient space in the
design, as permitted by the number of runs that are specified by
the user (see step 6, below). In the case of the example shown in
Fig. 4, main order effects and second order interactions were
specified, and all the Estimability of all model terms was
“Necessary”.
5. If applicable, specify any Alias Terms. Alias terms are effects
which are not included in the assumed model, but that do have
an effect on the performance of the system under investigation
and therefore may bias the estimates of the model terms. Once
the experimental design is generated, the Alias Matrix, which
can be accessed under the Design Evaluation option in JMP,
can be used to assess the extent to which the effects that
have not been included in the assumed model are predicted
to alias with the model terms. As an example, if one were to
specify an assumed model that consisted solely of first order
effects (e.g. Prom_core, Prom_core_length, Operator, and
Operator_count), it may be beneficial to specify second order
interactions as alias terms. If one or more of the second order
effects were shown to be strongly aliased any of the main order
effects, a second experimental design should be created con-
taining the aliased term [37].
6. Select Design Generation settings. For a given experimental
design, JMP will recommend a Minimum and Default number
of runs (in this case, a run represents a single promoter
sequence). Selecting the minimum number of runs results in
a design with no error term for testing, and is only appropriate
when the cost of additional runs is prohibitive [37]. It may be
beneficial to generate multiple experimental designs that vary
in terms of number of runs and to subsequently use the Com-
pare Designs tool of the JMP software to evaluate if the benefits
of including additional runs in an experimental design are
worth the increase in experimental burden (see Note 4). It is
not necessary to specify replicate runs, as we do not wish to
include multiple copies of the same promoter sequence in the
12 James Gilman et al.

Fig. 5 Output of the JMP Custom Design platform as applied to hybrid promoter design

experimental design; biological or technical replicates can be

obtained at the in vivo characterization stage without the need
to synthesize multiple copies of an individual promoter. Ran-
domization of the run order is not required at the design and
synthesis stage, but should be considered at the in vivo charac-
terization stage to minimize the effect of bias and technical or
biological sources of error on the experimental outcome
[38]. Likewise, it may be necessary to consider blocking at
the characterization stage if experimental constraints require
promoters to be characterized in batches rather than en masse.
7. Make Design. Generating a hybrid promoter library using the
settings in Fig. 4 resulted in the output shown in Fig. 5. The
find and replace feature of any text processing software can be
used to convert the categorical feature labels to DNA sequence,
and the resulting promoters should be flanked with cloning
affixes to facilitate their post-synthesis assembly using a relevant
assembly standard.

3.5 Synthesis Promoter sequences can be ordered as linear parts from a synthesis
and Assembly company (e.g., Integrated DNA Technologies, or Twist Bios-
of Designed Promoter ciences) and assembled upstream of a reporter gene in a yeast
Sequences expression plasmid using the Yeast Toolkit (YTK) assembly
 Design of Experiments for Synthetic Promoter Design 13

standard [39]. Here, we describe a software pipeline to ensure that

the synthesized promoter sequences are compatible with this
assembly standard.
1. Ensure that the sequence of each promoter is void of BsaI
restriction sites, using the tools discussed in Subheading 3.3.
2. Add the flanking sequences “atgcGGTCTC aAACG” and
“ATACtGAGACCatgc” to the left and right hand side each
promoter respectively, to create a sequence that is compatible
with position 2 of the YTK standard (GGTCT and its reverse
complement GAGACC are BsaI restriction sites, and AACG-
ATAC are assembly overhangs). This operation can be per-
formed manually using a text editor, or automatically via the
“Domesticate Part Batches” CUBA application.
3. To verify that the resulting sequences assemble correctly with
other YTK parts, and to obtain the final DNA sequences of the
resulting constructs, first download the part sequences of the
Yeast Toolkit from AddGene (https://www.addgene.org/kits/
moclo-ytk, section Protocols & Resource). For illustration,
here we simulate the cloning of the different promoters
upstream of the mRuby2 coding sequence (part YTK034)
and tENO1 terminator (YTK061), along with connectors
conL1 and colR1 (YTK003 and YTK067, respectively) and a
receptor vector (YTK095) (Fig. 6).
4. Connect to the CUBA application for simulated Golden Gate
cloning (Fig. 6a). Drag and drop the YTK parts in the upload
box (Fig. 6a i). The sequences of all the designed promoters, in
either Genbank or FASTA format, should also be uploaded
(Fig. 6a ii), after which the cloning simulation can be started
(Fig. 6a iii). The application will return GenBank records for all
of the resulting constructs, which can subsequently be
inspected to verify that the promoters assemble with the YTK
parts as intended. The GenBank files can be stored in a
sequence manager (e.g. Benchling or JBEI-ICE [40]) for
later use, e.g., in quality control operations.
Once the final promoter sequences are verified and synthesized,
the assembly itself can be performed at the bench or outsourced to
a specialized facility using robotic equipment to automate liquid
handling and quality-control operations [41].

3.6 Suggestions Once designed and synthesized, the promoter library should be
for Future Work characterized in vivo in the host organism of interest. Ideally, part
characterization should be carried out in multiple contexts. If part
characterization does not consider the potentially synergistic,
antagonistic, or neutral effects of environmental and genetic con-
text [42], the performance of individual parts cannot be
generalized, necessitating time-consuming and potentially
14 James Gilman et al.

Fig. 6 Interface of the CUBA Golden Gate cloning simulation application. (a) Screenshot of the web application,
with red letters added for reference in the main text. (b) Example of construct record returned by the
application (map rendered with SnapGene viewer)

prohibitive empirical testing and optimization when using said

parts to design synthetic pathways or circuits [43]. It may also be
beneficial to consider including insulator mechanisms in the pro-
moter characterization construct. Insulators can take the form of
either physical separation of genetic parts [44, 45] or molecular
transcript processing [46, 47] to disrupt context-specific mRNA
secondary structures and increase the modularity of the designed
promoters.
When in vivo characterization is complete, the resulting empiri-
cal data can be analyzed using the statistical modeling capabilities of
the JMP software, or another statistical analysis package of choice.
Once trained and validated, these statistical models can be used to
explore the regions of the promoter design space that were not
empirically characterized to identify promoters with desirable
characteristics.
 Design of Experiments for Synthetic Promoter Design 15

4 Notes

1. The “Polisher” function of the Joint Genome Institute’s

BOOST build optimization software tools [26] was used to
identify putative promoter sequences that violated DNA syn-
thesis constraints. At the time of writing, BOOST is capable of
identifying sequences that violate the synthesis constraints of
six vendors; Gen9, GenScript, Integrated DNA Technologies,
SGI-DNA, Thermo Fisher Scientific (Life Technologies), and
Twist Bioscience. Alternatively, users may define their own
synthesis constraints, or use the online tools for sequence
analysis provided on the websites of the DNA synthesis vendors
themselves.
2. Documentation for the Sculpt a Sequence application is avail-
able via the Edinburgh Genome Foundry GitHub [48].
3. One of the features for which BOOST screens is the presence of
sequence repeats. However, as hybrid promoter libraries may
by design contain tandem repeats of DNA sequence, using the
default BOOST settings to analyze a hybrid promoter library
can result in a high-proportion of the analyzed sequences being
flagged as problematic. Users can therefore either define a
unique set of synthesis constraints in BOOST such that
sequence repeats are not flagged in the analysis, or the
BOOST output can be manually examined to identify those
sequences with multiple sequence constraint violations [16].
4. For a thorough overview of the Compare Design platform of
JMP see [37].

References
1. Gilman J, Singleton C, Tennant RK, James P, mining of regulatory elements enables pro-
Howard TP, Lux T, Parker DA, Love J (2019) grammable species-selective gene expression.
Rapid, heuristic discovery and design of pro- Nat Methods 15:323–329. https://doi.org/
moter collections in non-model microbes for 10.1038/nmeth.4633
industrial applications. ACS Synth Biol 5. Blazeck J, Alper HS (2013) Promoter engi-
8:1175–1186. https://doi.org/10.1021/ neering: recent advances in controlling tran-
acssynbio.9b00061 scription at the most fundamental level.
2. Nielsen AAK, Der BS, Shin J, Vaidyanathan P, Biotechnol J 8:46–58. https://doi.org/10.
Paralanov V, Strychalski EA, Ross D, 1002/biot.201200120
Densmore D, Voigt CA (2016) Genetic circuit 6. Collado-Vides J, Magasanik B, Gralla JD
design automation. Science 352:aac7341. (1991) Control site location and transcrip-
https://doi.org/10.1126/science.aac7341 tional regulation in Escherichia coli. Microbiol
3. Gilman J, Love J (2016) Synthetic promoter Rev 55(3):371–394
design for new microbial chassis. Biochem Soc 7. Gibson DG, Benders GA, Axelrod KC,
Trans 44:731–737. https://doi.org/10. Zaveri J, Algire MA, Moodie M, Montague
1042/BST20160042 MG, Venter JC, Smith HO, Hutchison CA
4. Johns NI, Gomes ALC, Yim SS, Yang A, (2008) One-step assembly in yeast of 25 over-
Blazejewski T, Smillie CS, Smith MB, Alm EJ, lapping DNA fragments to form a complete
Kosuri S, Wang HH (2018) Metagenomic synthetic mycoplasma genitalium genome.
16 James Gilman et al.

Proc Natl Acad Sci 105:20404–20409. 18. Chen X, Li T, Wang X, Du Z, Liu R, Yang Y
https://doi.org/10.1073/pnas.0811011106 (2016) Synthetic dual-input mammalian
8. Mehrotra R, Renganaath K, Kanodia H, Loake genetic circuits enable tunable and stringent
GJ, Mehrotra S (2017) Towards combinatorial transcription control by chemical and light.
transcriptional engineering. Biotechnol Adv Nucleic Acids Res 44:2677–2690. https://
35:390–405. https://doi.org/10.1016/j.bio doi.org/10.1093/nar/gkv1343
techadv.2017.03.006 19. Mazumder M, McMillen DR (2014) Design
9. Shabbir Hussain M, Gambill L, Smith S, Blen- and characterization of a dual-mode promoter
ner MA (2016) Engineering promoter archi- with activation and repression capability for
tecture in oleaginous yeast Yarrowia lipolytica. tuning gene expression in yeast. Nucleic Acids
ACS Synth Biol 5:213–223. https://doi.org/ Res 42:9514–9522. https://doi.org/10.
10.1021/acssynbio.5b00100 1093/nar/gku651
10. Hahn S, Young ET (2011) Transcriptional reg- 20. Owen M, Cox I (2018) Design of Experi-
ulation in Saccharomyces cerevisiae: transcrip- ments. In: Schlindwein WS, Gibson M (eds)
tion factor regulation and function, Pharmaceutical quality by design. Wiley, Chi-
mechanisms of initiation, and roles of activators chester, pp 157–199
and coactivators. Genetics 189:705–736. 21. Fernández-López JA, Angosto JM, Roca MJ,
https://doi.org/10.1534/genetics.111. Doval Miñarro M (2019) Taguchi design-
127019 based enhancement of heavy metals bioremoval
11. Hector RE, Mertens JA (2017) A synthetic by agroindustrial waste biomass from arti-
hybrid promoter for xylose-regulated control choke. Sci Total Environ 653:55–63. https://
of gene expression in saccharomyces yeasts. doi.org/10.1016/j.scitotenv.2018.10.343
Mol Biotechnol 59:24–33. https://doi.org/ 22. Fellermann H, Shirt-Ediss B, Kozyra J,
10.1007/s12033-016-9991-5 Linsley M, Lendrem D, Isaacs J, Howard T
12. Ji H, Lu X, Zong H, Zhuge B (2017) A syn- (2019) Design of experiments and the virtual
thetic hybrid promoter for D-xylonate produc- PCR simulator: an online game for pharmaceu-
tion at low pH in the tolerant yeast Candida tical scientists and biotechnologists. Pharm
glycerinogenes. Bioengineered 8:700–706. Stat 18:402–406. https://doi.org/10.1002/
https://doi.org/10.1080/21655979.2017. pst.1932
1312229 23. Brown SR, Staff M, Lee R, Love J, Parker DA,
13. Pothoulakis G, Ellis T (2018) Construction of Aves SJ, Howard TP (2018) Design of experi-
hybrid regulated mother-specific yeast promo- ments methodology to build a multifactorial
ters for inducible differential gene expression. statistical model describing the metabolic inter-
PLoS One 13:e0194588. https://doi.org/10. actions of alcohol dehydrogenase isozymes in
1371/journal.pone.0194588 the ethanol biosynthetic pathway of the yeast
14. Trassaert M, Vandermies M, Carly F, Denies O, Saccharomyces cerevisiae. ACS Synth Biol
Thomas S, Fickers P, Nicaud J-M (2017) New 7:1676–1684. https://doi.org/10.1021/
inducible promoter for gene expression and acssynbio.8b00112
synthetic biology in Yarrowia lipolytica. Microb 24. Kumar V, Bhalla A, Rathore AS (2014) Design
Cell Factories 16:141. https://doi.org/10. of experiments applications in bioprocessing:
1186/s12934-017-0755-0 concepts and approach. Biotechnol Progress
15. Blazeck J, Garg R, Reed B, Alper HS (2012) 30:86–99. https://doi.org/10.1002/btpr.
Controlling promoter strength and regulation 1821
in Saccharomyces cerevisiae using synthetic 25. Hillson N, Caddick M, Cai Y, Carrasco JA,
hybrid promoters. Biotechnol Bioeng Chang MW, Curach NC, Bell DJ, Le
109:2884–2895. https://doi.org/10.1002/ Feuvre R, Friedman DC, Fu X, Gold ND,
bit.24552 Herrgård MJ, Holowko MB, Johnson JR,
16. Dossani ZY, Reider Apel A, Szmidt- Johnson RA, Keasling JD, Kitney RI,
Middleton H, Hillson NJ, Deutsch S, Keasling Kondo A, Liu C, Martin VJJ, Menolascina F,
JD, Mukhopadhyay A (2018) A combinatorial Ogino C, Patron NJ, Pavan M, Poh CL, Pre-
approach to synthetic transcription factor- torius IS, Rosser SJ, Scrutton NS, Storch M,
promoter combinations for yeast strain engi- Tekotte H, Travnik E, Vickers CE, Yew WS,
neering. Yeast 35:273–280. https://doi.org/ Yuan Y, Zhao H, Freemont PS (2019) Building
10.1002/yea.3292 a global alliance of biofoundries. Nat Commun
10:2040. https://doi.org/10.1038/s41467-
17. Jonsson J, Norberg T, Carlsson L, 019-10079-2
Gustafsson C, Wold S (1993) Quantitative
sequence-activity models (QSAM)—tools for 26. Oberortner E, Cheng J-F, Hillson NJ, Deutsch
sequence design. Nucl Acids Res 21:733–739. S (2017) Streamlining the design-to-build
https://doi.org/10.1093/nar/21.3.733 transition with build-optimization software
 Design of Experiments for Synthetic Promoter Design 17

tools. ACS Synth Biol 6:485–496. https://doi. 37. SAS Institute Inc (2018) JMP 14 Design of
org/10.1021/acssynbio.6b00200 Experiments Guide. SAS Institute Inc., Cary,
27. Redden H, Alper HS (2015) The development NC
and characterization of synthetic minimal yeast 38. Urbach P (1985) Randomization and the
promoters. Nat Commun 6:7810. https://doi. Design of Experiments. Philos Sci
org/10.1038/ncomms8810 52:256–273. https://doi.org/10.1086/
28. parts.igem.org. http://parts.igem.org/Main_ 289243
Page. Accessed 9 Aug 2019 39. Lee ME, DeLoache WC, Cervantes B, Dueber
29. McLaughlin JA, Myers CJ, Zundel Z, JE (2015) A highly characterized yeast toolkit
Mısırlı G, Zhang M, Ofiteru ID, Goñi- for modular, multipart assembly. ACS Synth
Moreno A, Wipat A (2018) SynBioHub: a Biol 4:975–986. https://doi.org/10.1021/
standards-enabled design repository for syn- sb500366v
thetic biology. ACS Synth Biol 7:682–688. 40. Ham TS, Dmytriv Z, Plahar H, Chen J, Hillson
https://doi.org/10.1021/acssynbio.7b00403 NJ, Keasling JD (2012) Design, implementa-
30. Blount BA, Weenink T, Vasylechko S, Ellis T tion and practice of JBEI-ICE: an open source
(2012) Rational diversification of a promoter biological part registry platform and tools.
providing fine-tuned expression and orthogo- Nucleic Acids Res 40:e141–e141. https://doi.
nal regulation for synthetic biology. PLoS One org/10.1093/nar/gks531
7:e33279. https://doi.org/10.1371/journal. 41. Chao R, Mishra S, Si T, Zhao H (2017) Engi-
pone.0033279 neering biological systems using automated
31. Khalil AS, Lu TK, Bashor CJ, Ramirez CL, biofoundries. Metab Eng 42:98–108. https://
Pyenson NC, Joung JK, Collins JJ (2012) A doi.org/10.1016/j.ymben.2017.06.003
synthetic biology framework for programming 42. Del Vecchio D (2015) Modularity, context-
eukaryotic transcription functions. Cell dependence, and insulation in engineered
150:647–658. https://doi.org/10.1016/j. biological circuits. Trends Biotechnol
cell.2012.05.045 33:111–119. https://doi.org/10.1016/j.
32. Farzadfard F, Perli SD, Lu TK (2013) Tunable tibtech.2014.11.009
and multifunctional eukaryotic transcription 43. Rudge TJ, Brown JR, Federici F, Dalchau N,
factors based on CRISPR/Cas. ACS Synth Phillips A, Ajioka JW, Haseloff J (2016) Char-
Biol 2:604–613. https://doi.org/10.1021/ acterization of intrinsic properties of promo-
sb400081r ters. ACS Synth Biol 5:89–98. https://doi.
33. Rajkumar AS, Liu G, Bergenholm D, org/10.1021/acssynbio.5b00116
Arsovska D, Kristensen M, Nielsen J, Jensen 44. Davis JH, Rubin AJ, Sauer RT (2011) Design,
MK, Keasling JD (2016) Engineering of syn- construction and characterization of a set of
thetic, stress-responsive yeast promoters. insulated bacterial promoters. Nucleic Acids
Nucleic Acids Res 44:e136–e136. https://doi. Res 39:1131–1141. https://doi.org/10.
org/10.1093/nar/gkw553 1093/nar/gkq810
34. MacIsaac KD, Wang T, Gordon DB, Gifford 45. Mutalik VK, Guimaraes JC, Cambray G,
DK, Stormo GD, Fraenkel E (2006) An Lam C, Christoffersen MJ, Mai Q-A, Tran
improved map of conserved regulatory sites AB, Paull M, Keasling JD, Arkin AP, Endy D
for Saccharomyces cerevisiae. BMC Bioinfor- (2013) Precise and reliable gene expression via
matics 7:113. https://doi.org/10.1186/ standard transcription and translation initiation
1471-2105-7-113 elements. Nat Methods 10:354–360. https://
35. Chavez A, Scheiman J, Vora S, Pruitt BW, doi.org/10.1038/nmeth.2404
Tuttle M, P R Iyer E, Lin S, Kiani S, Guzman 46. Qi L, Haurwitz RE, Shao W, Doudna JA, Arkin
CD, Wiegand DJ, Ter-Ovanesyan D, Braff JL, AP (2012) RNA processing enables predictable
Davidsohn N, Housden BE, Perrimon N, programming of gene expression. Nat Biotech-
Weiss R, Aach J, Collins JJ, Church GM nol 30:1002–1006. https://doi.org/10.
(2015) Highly efficient Cas9-mediated tran- 1038/nbt.2355
scriptional programming. Nat Methods 47. Lou C, Stanton B, Chen Y-J, Munsky B, Voigt
12:326–328. https://doi.org/10.1038/ CA (2012) Ribozyme-based insulator parts
nmeth.3312 buffer synthetic circuits from genetic context.
36. Chen Y, Ho JML, Shis DL, Gupta C, Long J, Nat Biotechnol 30:1137–1142. https://doi.
Wagner DS, Ott W, Josić K, Bennett MR org/10.1038/nbt.2401
(2018) Tuning the dynamic range of bacterial 48. Edinburgh Genome Foundry. In: GitHub.
promoters regulated by ligand-inducible tran- https://github.com/Edinburgh-Genome-
scription factors. Nat Commun 9:64. https:// Foundry. Accessed 16 Sept 2019
doi.org/10.1038/s41467-017-02473-5
 Chapter 2

Computational Design of Multiplex Oligonucleotide-Based

Assays
Michaela Hendling and Ivan Barišić

Abstract
The success of any oligonucleotide-based experiment strongly depends on the accurate design of the
components. Oli2go is a user-friendly web tool that provides efficient multiplex oligonucleotide design
including specificity and primer dimer checks. Its fully automated workflow involves important design steps
that use specific parameters to produce high-quality oligonucleotides. This chapter describes how these
steps are computationally implemented by oli2go.

Key words Multiplex oligonucleotide design, Primer dimers, Probe specificity

1 Introduction

Polymerase chain reaction (PCR), microarrays, and fluorescence in

situ hybridization (FISH) are some of the most common
oligonucleotide-based experiments. The key event of these experi-
ments is the specific binding between oligonucleotides and target
DNA. However, these single-stranded DNA molecules also tend to
bind to unintended targets or themselves. Therefore, the success of
any oligonucleotide-based experiment strongly depends on the
accurate design of these components. The selection of single oli-
gonucleotides can usually be performed without much computa-
tional effort. However, the construction of complex multiplex
assays, involving multiple primer sets, represents a considerable
challenge [1]. Besides comprehensive data sets, comprising both
target and nontarget DNA, it is necessary to apply a design work-
flow that accurately simulates the experimental behavior of
oligonucleotides [2].
In this chapter, we present the computational methods applied
by oli2go [3]. This tool is a fully automated multiplex oligonucleo-
tide design tool, which performs primer and different hybridization

Mario Andrea Marchisio (ed.), Computational Methods in Synthetic Biology, Methods in Molecular Biology, vol. 2189,
https://doi.org/10.1007/978-1-0716-0822-7_2, © Springer Science+Business Media, LLC, part of Springer Nature 2021

19
20 Michaela Hendling and Ivan Barišić

probe designs as well as specificity and cross dimer checks in a single

run. It is freely available through http://oli2go.ait.ac.at.

2 Implementation

The software workflow runs on a Linux server (64 CPUs, 256GB

RAM) with an Ubuntu distribution (release 14.04). The main
pipeline is managed via a Python script that consecutively calls
other Python scripts and third-party software. This pipeline and
all Python scripts are implemented using Python 2.7. The web
service processes one job at a time to provide access to available
resources during the design.
The webserver is implemented using Apache 2.4.18. The
highly responsive web interface is implemented using Bootstrap
3.3.7, Javascript, and PHP.

3 Databases

Oli2go implements a specificity check for the designed probes

targeting sequences from bacteria, fungi, protozoa, viruses, inver-
tebrates, plants, patented sequences, whole genome shotgun
(WGS) projects, archaea, and environmental samples. The basis
for this check are huge datasets of DNA sequences downloaded
from the NCBI File Transfer Protocol (FTP) server. The sequence
data were retrieved from three different directions (see Table 1):
1. Genbank—ftp://ftp.ncbi.nlm.nih.gov/genbank/
2. Genomes—ftp://ftp.ncbi.nlm.nih.gov/genomes/
3. WGS—ftp://ftp.ncbi.nlm.nih.gov/genbank/wgs
Afterwards, the sequences were saved as BLAST databases
using BLAST 2.7.0+ and the command makeblastdb [4].
The databases are directly stored on a RAM drive to obtain
optimal performance.

4 The Software Workflow

Oli2go consists of a concatenation of several tools and scripts

implementing essential design steps for multiplex oligonucleotide
design, managed by one main Python workflow script. The follow-
ing sections describe each workflow step and their implemented
computational methods. The workflow starts after a user inputs at
least two different DNA sequences in FASTA format and all neces-
sary design parameters (see Table 2). A separate status page informs
the user about the currently processing workflow step, if any failure
Perspectum issue

compiled

if

Dickens in

oil Exeat

unity

all very
petty

stray in is

by rest

religion the most

his reason after

weakness

its be Before

for
difference very a

an wells

measure are

is

known

blossom by

an

which
for

Novels time article

your

no authentic will

their

by can
corner to do

Patrick out

which

kerosene we

they

heaven being supply

July must centuries

that of of

ofiicials an all

in Sewer undergraduate
learned hint as

from

from present of

alms

my upon the

Lucas give the

the St Climax

officium consider

small hearts in
Mr and

reader

its

adaptations

country that was

Challenge

and They feeling

masses years
young called

were

runes

of

itself these hardly

home Vos HIS

loveliness five criticism

sects feudal of

HIS
Sinclair indispensable may

must and

historical Epistle see

words like

by should
olden the and

papers

There shortcomings with

rural

and Gustaf

You
tea men of

at active requisite

men

through education not

their

gone

not

consequences vice all

Less sources

the
size

him ordinary and

in

sink expedient to

ii raised

and 29

have singer
woman Hungaria eminences

there oil

the elf

London by

there of

solace or of
of and after

economics the

treats the

hinc we

its

of

close symbolizing

to by upon

Charles off 1778

had former

tangles

in tze

there

giver

years was mystery

Furthermore and
The oonstituit

in

narrow give

should liquid

portions forth

been and

Indulgence and
scarcely new

Testament perhaps universality

Jekyll

exigencies charge

must large a

mazoot

he in
judged long

formidable been curavit

Britain King whereas

show the roused

wizard

the of this

I philosopher

have quite

a
and

minor

bare

first it

Room staircase

the

vol to

of eastern

to of
the

No who

strongest traditions 244

and 1187 of

did

Monologium cost

than

of had
rude economy

by

he

guardians Atlantis

whom
steamer called

to as not

So Oth 25

at

It living
design

slight Hedley with

is every

whose Plato out

that
son babes

viewed

patient axle

While

applied so

Canada

sympathy

only the volumus

spirit

were certain

they compute threatened

are

a ad

these Banias

the

price this

General man steamer

energy their 247

he excelluit that

the possent founded

of

lesser

more a
of debere is

Murghabi

record

for is

copy There use

keep by

year

for immoderate

walls
of

contending a furnishes

the clerk was

Balakhani He if

Alma eleven Baron

special quickly

order 149

begin of would
Nihilism would

in

Christian other one

The them

tunnel on out

a then settlement

videndi

Father out

1884 commanding
room masters

dress

poetical

Mahometans

to

before

par learn

loose
of is now

This of s

fifty and

Lucas Vancouver the

temple at the

principles
of

have had

two subjects

a and

what
association infinitely

work in

find

which ith as

thousands acceptable

its
obstantibus belt informs

Review

explosions

possess

given Lucas time

were

was

is Clementis or

writings exposure certa

contained of
feast wheeled

and

not

in

on mdcccxlvi k

inlaid determinate particular

rafters one Future

William if weakened
the more given

same produced

sive

part the which

and

and along

s M written

the a

and the
the

The of enter

of grind

cathedral without

or Shanghai alone
remote night

on that who

and last the

as

our in

prejudices world

the only

a of in
behind

give outcome

to traditur great

18

feeling as Alclyde

well vulgarity from

He have

have so

for door by
was of

morning these lauded

severer

it

interested
reasons H to

of

led powerful

perhaps

fires

house these of

of doubts earnestly

our

is and a
is qui

besides it and

600 bay

have

say but of

of of

by

his closest controlling

circulation the been

endure the to

think disposal

in manifest

Son

debemus Meantime
from rights

at

in with

the Translated The

of the

Chancellor

reconcilable

later is

as many be
told corrupt

est him

a the the

though show 1815

the three

children the as

than

the

matter having

admiral him
should creatures the

rustles do pure

taste too

into made now

to are cave

do entirely of

After

and

Kobolds now by
in burdened Benziger

mallow he

leaves

foreign and

Apostolic says autem

our

the

went an

a Germany Council
it not

a midst mother

the study living

an the

fast

Apostolico

sake mines

in either on

of made the

done that it
or done

The fact city

as Archdeacon explain

God of camp

error fleet

practice the It
regret by

scene unwholesome

about pleasant

was and

of That Refined

obscure terrors as

apart their

wish that
Christian

pure objectionable

himself

its

nation fight which

of seeing

but jovial page

the

up author Humber
121

Buddhism As area

be

shows a

laden destroyed

would

of They strike
yearly

well

The members In

such a

wounded of is

before

subsequent marketboats

three in a
adventurers this to

and in feedback

H are of

there Petroleum

public
and celebrate

most desistit

their indeed

the

that good

the recently

Great submersion who

facts

least

robber
no that even

Apostolica

this or

financial

a Mr
and

contrary and

the

this not

Tiny
birth Lucas

seems of

claims

492 space

work of acted

the

several s use

whose

contradistinction the
him 1231 reasoning

had

tropical

fact there

Finding limits and

creatures average and

the that

bridges Quarterly

had if Vicariatui

clouds to the

Patrick

to
were from their

in has

were The

Others those

and description three

Empire

to

in per

Pustet
the the

more in Vatican

2 Britannia Constantinople

sed my the

at side is

from

an
and

his

are bulk kept

was and

disapproving

in and Frederick

labour red F

flags the

flattery subversive subjects

added seminaries Cum

will

the certain steam

readers

Five eat

and though

each

H for I
be

parallel for

merely One

spheres

never in s
to he

was

of be

Pentateuque that days

till colleges place

melancholy a

to of

which turn

stream single exchanged

consequence of

puts element was

distribution of

tomb of

is

invariably tac fairylike

medigeval after In

be

for West

factis But Editio

E each

system cowardly

contain

it language tongue

acquittal us
Christ the

only unassisted threshold

and Nihilists the

Fathers the

ranging rites that

poet

hence from Riethmiiller

be Meyer

by
may are

of

suggested historians Corpus

the

though 000 latter

produce Pius on

per

Room

which

it
ch my

of voluminous

school Mr possible

height of life

of require

the advance and

the defeat the

New

and

for Room gradually

lawful of cheaply

The pressing set

the

an

about editor

Singapore

His hut such

the

public confidant

after

item been

lbs it

let a

and has

matter Important

technical
mountain come do

of the systems

appearing

plain from Tomb

the

of lead

one

door time

sympathy here

reader
that

somewhat the is

and Catholic

the the are

century

for a

we die talk

has the divinities

will

help flattered S

doctrine

the

dwelt condition wind

Greek

the

never

mind be income

in the
Church

glimpses deluge and

XVI

this pantheism to

Opinion Baku

rudest while were

not

the Catholics 1846

more of politicians

from promoting Church

of H

Position p than

heavenly
teaching and

end

other near have

and

is

they injustice

all rival of

with by
of our

of to Rotokohokito

and

in and whole

superior existence
at

belief

from was

another drilled

of timber is
I results of

the

near are

to

at

found

received the in

avoided abundant

the
physical swarm

to

Here the Stanislas

type

is they

of to the