Methods in
Molecular Biology 2760
Jeffrey Carl Braman Editor
Synthetic
Biology
Methods and Protocols
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.
,Synthetic Biology
Methods and Protocols
Second Edition
Edited by
Jeffrey Carl Braman
Agilent Technologies, Inc, La Jolla, CA, USA
,Editor
Jeffrey Carl Braman
Agilent Technologies, Inc
La Jolla, CA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-0716-3657-2 ISBN 978-1-0716-3658-9 (eBook)
https://doi.org/10.1007/978-1-0716-3658-9
© The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part
of Springer Nature 2024
This work is subject to copyright. All rights are solely and exclusively licensed 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.
Paper in this product is recyclable.
,Preface
Synthetic Biology represents a unique branch of science requiring the expertise of chemists,
biologists, and engineers. This broad statement is intended to entice biochemists, analytical
and organic chemists, chemical engineers, molecular biologists, computer scientists, soft-
ware, mechanical, and electrical engineers, biophysicists, plant and evolutionary biologists,
process and manufacturing engineers, and pharmaceutical scientists into research programs
and collaborations that will solve difficult problems such as producing renewable energy
resources, feeding an ever-increasing world population, and curing disease, to name just a
few examples.
The second edition of Synthetic Biology complements and enhances the first edition and
provides you with updated and enhanced techniques and tools. As with the first edition,
experienced contributors to the second edition have previously published their work in
reputable, peer-reviewed periodicals, and their contributions to this second edition provide
expert step-by-step guidance and ideas for conducting your own synthetic biology research.
La Jolla, CA, USA Jeffrey Carl Braman
v
,Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
PART I GENE CIRCUITS AND BIOCHEMICAL PATHWAYS
1 Plant Engineering to Enable Platforms for Sustainable Bioproduction
of Terpenoids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Jacob D. Bibik and Björn Hamberger
2 Compartmentalized Terpenoid Production in Plants
Using Agrobacterium-Mediated Transient Expression . . . . . . . . . . . . . . . . . . . . . . . 21
Jacob D. Bibik, Abigail E. Bryson, and Björn Hamberger
3 Design Principles for Biological Adaptation: A Systems and Control-Theoretic
Treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Priyan Bhattacharya, Karthik Raman, and Arun K. Tangirala
4 Construction of Xylose-Utilizing Cyanobacterial Chassis for Bioproduction
Under Photomixotrophic Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Xinyu Song, Yue Ju, Lei Chen, and Weiwen Zhang
5 Allosteric-Regulation-Based DNA Circuits in Saccharomyces cerevisiae
to Detect Organic Acids and Monitor Hydrocarbon Metabolism In Vitro . . . . . 77
Michael Dare Asemoloye and Mario Andrea Marchisio
6 dCas12a:Pre-crRNA: A New Tool to Induce mRNA Degradation
in Saccharomyces cerevisiae Synthetic Gene Circuits . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Lifang Yu and Mario Andrea Marchisio
PART II GENOME EDITING AND MODIFICATION
7 CRISPRi-Driven Genetic Screening for Designing Novel
Microbial Phenotypes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Minjeong Kang, Kangsan Kim, and Byung-Kwan Cho
8 Enzymatic Preparation of DNA with an Expanded Genetic Alphabet
Using Terminal Deoxynucleotidyl Transferase and Its Applications. . . . . . . . . . . . 133
Guangyuan Wang, Yuhui Du, and Tingjian Chen
9 Single-Nucleotide Microbial Genome Editing Using CRISPR-Cas12a. . . . . . . . . 147
Ho Joung Lee and Sang Jun Lee
10 Multiplex Marker-Less Genome Integration in Pichia pastoris
Using CRISPR/Cas9 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Jucan Gao, Jintao Cheng, and Jiazhang Lian
vii
,viii Contents
11 Genome Editing, Transcriptional Regulation, and Forward Genetic Screening
Using CRISPR-Cas12a Systems in Yarrowia lipolytica. . . . . . . . . . . . . . . . . . . . . . . 169
Adithya Ramesh, Sangcheon Lee, and Ian Wheeldon
12 An Improved Method for Eliminating or Creating Intragenic
Bacterial Promoters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Ellina Trofimova, Dominic Y. Logel, and Paul R. Jaschke
13 Genetic Code Expansion in Pseudomonas putida KT2440. . . . . . . . . . . . . . . . . . . . 209
Tianyu Gao, Jiantao Guo, and Wei Niu
14 Genome-Wide Screen for Enhanced Noncanonical Amino Acid
Incorporation in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
Briana R. Lino and James A. Van Deventer
15 Positive Selection Screens for Programmable Endonuclease Activity
Using I-SceI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Michael A. Mechikoff, Kok Zhi Lee, and Kevin V. Solomon
16 CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa . . . . . . . . . . 267
Giulia Ravagnan, Meliawati Meliawati, and Jochen Schmid
PART III GENOME LANGUAGE AND COMPUTING
17 Programming Juxtacrine-Based Synthetic Signaling Networks
in a Cellular Potts Framework . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
Calvin Lam and Leonardo Morsut
18 Encoding Genetic Circuits with DNA Barcodes Paves the Way
for High-Throughput Profiling of Dose-Response Curves
of Metabolite Biosensors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
Huibao Feng, Yikang Zhou, and Chong Zhang
19 Machine Learning for Biological Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319
Tom Blau, Iadine Chades, and Cheng Soon Ong
20 A Machine Learning Approach for Predicting Essentiality
of Metabolic Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345
Lilli J. Freischem and Diego A. Oyarzún
21 The Causes for Genomic Instability and How to Try and Reduce
Them Through Rational Design of Synthetic DNA . . . . . . . . . . . . . . . . . . . . . . . . . 371
Matan Arbel-Groissman, Itamar Menuhin-Gruman,
Hader Yehezkeli, Doron Naki, Shaked Bergman,
Yarin Udi, and Tamir Tuller
22 Genetic Network Design Automation with LOICA . . . . . . . . . . . . . . . . . . . . . . . . . 393
Gonzalo Vidal, Carolus Vitalis, Tamara Matúte,
Isaac Núñez, Fernán Federici, and Timothy J. Rudge
23 Flapjack: Data Management and Analysis for Genetic
Circuit Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413
Carolus Vitalis, Guillermo Yáñez Feliú, Gonzalo Vidal,
Macarena Muñoz Silva, Tamara Matúte, Isaac Núñez,
Fernán Federici, and Timothy J. Rudge
, Contents ix
PART IV MOLECULAR ASSEMBLY
24 In Vivo DNA Assembly Using the PEDA Method . . . . . . . . . . . . . . . . . . . . . . . . . . 437
Tianyuan Su, Qingxiao Pang, and Qingsheng Qi
25 Cell-Free Synthesis and Quantitation of Bacteriophages . . . . . . . . . . . . . . . . . . . . . 447
Antoine Levrier, Steven Bowden, Bruce Nash,
Ariel Lindner, and Vincent Noireaux
26 Multimodal Control of Bacterial Gene Expression by Red
and Blue Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
Stefanie S. M. Meier, Elina Multam€ a ki, Américo T. Ranzani,
Heikki Takala, and Andreas Möglich
27 In Silico Design, In Vitro Construction, and In Vivo Application
of Synthetic Small Regulatory RNAs in Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . 479
Michel Brück, Bork A. Berghoff, and Daniel Schindler
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
, Contributors
MATAN ARBEL-GROISSMAN • Shmunis School of Biomedicine and Cancer Research, The George
S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel
MICHAEL DARE ASEMOLOYE • School of Pharmaceutical Science and Technology, Tianjin
University, Tianjin, China
BORK A. BERGHOFF • Institute for Microbiology and Molecular Biology, Justus-Liebig
University Giessen, Giessen, Germany
SHAKED BERGMAN • Department of Biomedical Engineering, Tel Aviv University, Tel Aviv,
Israel
PRIYAN BHATTACHARYA • Department of Chemical Engineering, Indian Institute of
Technology, Madras (IIT Madras), Chennai, India; Robert Bosch Centre of Data Science
and Artificial Intelligence (RBCDSAI), IIT Madras, Chennai, India; Initiative for
Biological Science and Systems mEdicine (IBSE), IIT Madras, Chennai, India
JACOB D. BIBIK • Department of Biochemistry, Michigan State University, East Lansing,
USA; MelaTech, LLC, Baltimore, Maryland, USA
TOM BLAU • CSIRO, Data 61, Eveleigh, NSW, Australia
STEVEN BOWDEN • University of Minnesota, Department of Food Science and Nutrition,
Saint Paul, MN, USA
MICHEL BRÜCK • Max Planck Institute for Terrestrial Microbiology, Marburg, Germany;
Institute for Microbiology and Molecular Biology, Justus-Liebig University Giessen, Giessen,
Germany
ABIGAIL E. BRYSON • Department of Biochemistry and Molecular Biology, Michigan State
University, East Lansing, MI, USA
IADINE CHADES • CSIRO, Environment, Brisbane, QLD, Australia
JINTAO CHENG • Key Laboratory of Biomass Chemical Engineering of Ministry of Education,
College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, China;
ZJU-Hangzhou Global Scientific and Technological Innovation Center, Zhejiang
University, Hangzhou, China
LEI CHEN • Laboratory of Synthetic Microbiology, School of Chemical Engineering &
Technology, Tianjin University, Tianjin, People’s Republic of China; Key Laboratory of
Systems Bioengineering and Frontier Science Center of Synthetic Biology, the Ministry of
Education of China, Tianjin University, Tianjin, People’s Republic of China
TINGJIAN CHEN • MOE International Joint Research Laboratory on Synthetic Biology and
Medicines, School of Biology and Biological Engineering, South China University of
Technology, Guangzhou, P. R. China
BYUNG-KWAN CHO • Department of Biological Sciences, Korea Advanced Institute of Science
and Technology, Daejeon, Republic of Korea; Innovative Biomaterials Research Center, KI
for the BioCentury, Korea Advanced Institute of Science and Technology, Daejeon, Republic
of Korea; Intelligent Synthetic Biology Center, Daejeon, Republic of Korea
YUHUI DU • MOE International Joint Research Laboratory on Synthetic Biology and
Medicines, School of Biology and Biological Engineering, South China University of
Technology, Guangzhou, P. R. China
FERNÁN FEDERICI • ANID-Millennium Science Initiative Program Millennium Institute for
Integrative Biology (iBio), Santiago, Chile; FONDAP Center for Genome Regulation,
xi
