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METHODS IN MOLECULAR BIOLOGY™

Series Editor
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School of Life Sciences
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 Gene Synthesis

Methods and Protocols

Edited by

Jean Peccoud
Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
Editor
Jean Peccoud, Ph.D.
Virginia Bioinformatics Institute
Virginia Tech
Blacksburg, VA, USA
[email protected]

ISSN 1064-3745 e-ISSN 1940-6029

ISBN 978-1-61779-563-3 e-ISBN 978-1-61779-564-0
DOI 10.1007/978-1-61779-564-0
Springer New York Dordrecht Heidelberg London

Library of Congress Control Number: 2012930137

© Springer Science+Business Media, LLC 2012

All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the
publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA),
except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information
storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or
hereafter developed is forbidden.
The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified
as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights.

Printed on acid-free paper

Humana Press is part of Springer Science+Business Media (www.springer.com)

Preface

The de novo fabrication of custom DNA molecules is a transformative technology that

significantly affects the biotechnology industry. Basic genetic engineering techniques for
manipulating DNA in vitro opened an incredible field of opportunity in the life sciences.
However, genetic engineering has now moved beyond the introduction of single genes into
cells to multigene cassettes, and is rapidly progressing toward whole genome engineering.
In this new context, the synthesis of DNA molecules has resurged as the time and cost-
limiting step in genetic engineering.
Today, most multigene engineering projects involve ad hoc methods of DNA assembly.
A variety of PCR-based methods are in common use alongside more traditional restriction
enzyme-based assembly methods. Their essential feature is the piecing together of existing
DNAs that are cloned from natural sources. These techniques present a number of limita-
tions. The use of restriction sites within natural sequences necessitates a labor intensive
custom cloning strategy that is difficult to automate. As a result, molecular biologists often
reach a tacit compromise between obtaining a desired sequence and the number of steps in
the cloning process they are willing or able to undertake in constructing it.
Theoretically, DNA fabrication methods that are rooted in chemical synthesis could
transform synthesis into a generic, predictable, and scalable process allowing the generation
of any user-defined DNA sequence. By liberating the process from the confines of preexisting
sequences, the problem of composition design becomes orthogonal to the problem of
physical construction. Therefore, as gene synthesis becomes a commodity, biologists will
spend more time designing custom DNA molecules and characterizing their performance,
and less time constructing them.
One day, DNA may be fabricated using a purely chemical process. Today, however,
DNA fabrication still involves sophisticated cloning techniques, but nevertheless a transi-
tion period has already emerged. Academic and commercial operators experiment with
complex processes that combine the assembly of chemically synthesized oligos with cloning
steps in attempts to construct long DNA molecules. Even though a number of companies
have rushed to and sometimes later walked away from the gene synthesis market, DNA
fabrication is not a black box that would involve radically different techniques than those
commonly used in a molecular biology laboratory, nor does it require expensive equip-
ment. Depending on the context it might make sense to outsource DNA fabrication to an
external vendor, but in other cases there might be value in performing part of the process
in house. In fact, gene synthesis projects are approachable by undergraduate students
enabled by straightforward protocols and training in a relatively small set of molecular biology
skills. In any case, it is important to understand that the fabrication of small DNA fragment
(less than 1 kb) is often very straightforward, but the assembly of longer DNA molecules
raises a number of inherent technical difficulties that need to be understood.

v
vi Preface

This book provides step-by-step protocols for the different stages of a DNA fabrication
process. Section I focuses on protocols used for the assembly of oligonucleotides in building
blocks also called synthons. The cloning of synthons into larger fragments up to the size of
bacterial genomes is the focus of Section II. Bioinformatics protocols and software applica-
tions necessary to design gene synthesis protocols are described in Section III. Finally,
Section IV describes the educational and biosecurity impacts of gene synthesis.
Any laboratory relying on recombinant DNA technology for its research is a potential
user of gene synthesis. Few laboratories will develop a completely home grown gene syn-
thesis process. Oligonucleotide synthesis or sequencing will most likely be outsourced to a
core facility or a commercial operator. In other cases, the synthesis of longer fragments may
also be outsourced. By providing step-by-step descriptions of all the different stages of a
complex gene synthesis process, this book will help readers refine their understanding of
gene synthesis and determine what part of the process they can or should do in their labora-
tory and what parts should be contracted to a specialized service provider.

Blacksburg, VA, USA Jean Peccoud, Ph.D.

Contents

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

PART I ASSEMBLY OF OLIGONUCLEOTIDES IN SYNTHONS

1 Building Block Synthesis Using the Polymerase

Chain Assembly Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Julie A. Marchand and Jean Peccoud
2 Oligonucleotide Assembly in Yeast to Produce
Synthetic DNA Fragments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Daniel G. Gibson
3 TopDown Real-Time Gene Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Mo Chao Huang, Wai Chye Cheong, Hongye Ye,
and Mo-Huang Li
4 De Novo DNA Synthesis Using Single-Molecule PCR . . . . . . . . . . . . . . . . . . 35
Tuval Ben Yehezkel, Gregory Linshiz, and Ehud Shapiro

PART II SYNTHON ASSEMBLY

5 SLIC: A Method for Sequence- and Ligation-Independent Cloning . . . . . . . . 51

Mamie Z. Li and Stephen J. Elledge
6 Assembly of Standardized DNA Parts Using BioBrick Ends in E. coli. . . . . . . . 61
Olivia Ho-Shing, Kin H. Lau, William Vernon, Todd T. Eckdahl,
and A. Malcolm Campbell
7 Assembling DNA Fragments by USER Fusion . . . . . . . . . . . . . . . . . . . . . . . . 77
Narayana Annaluru, Héloïse Muller, Sivaprakash Ramalingam,
Karthikeyan Kandavelou, Viktoriya London, Sarah M. Richardson,
Jessica S. Dymond, Eric M. Cooper, Joel S. Bader, Jef D. Boeke,
and Srinivasan Chandrasegaran
8 Fusion PCR via Novel Overlap Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
Kamonchai Cha-aim, Hisashi Hoshida, Tomoaki Fukunaga,
and Rinji Akada
9 Using Recombineering to Generate Point Mutations:
The Oligonucleotide-Based “Hit and Fix” Method . . . . . . . . . . . . . . . . . . . . . 111
Suhwan Chang, Stacey Stauffer, and Shyam K. Sharan
10 Using Recombineering to Generate Point Mutations:
galK-Based Positive–Negative Selection Method . . . . . . . . . . . . . . . . . . . . . . . 121
Kajal Biswas, Stacey Stauffer, and Shyam K. Sharan

vii
viii Contents

11 Assembling Large DNA Segments in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . 133

Héloïse Muller, Narayana Annaluru, Joy Wu Schwerzmann,
Sarah M. Richardson, Jessica S. Dymond, Eric M. Cooper,
Joel S. Bader, Jef D. Boeke, and Srinivasan Chandrasegaran
12 Recursive Construction of Perfect DNA Molecules and Libraries
from Imperfect Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
Gregory Linshiz, Tuval Ben Yehezkel, and Ehud Shapiro
13 Cloning Whole Bacterial Genomes in Yeast . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Gwynedd A. Benders
14 Production of Infectious Poliovirus from Synthetic Viral Genomes . . . . . . . . . 181
Jeronimo Cello and Steffen Mueller

PART III SOFTWARE FOR GENE SYNTHESIS

15 In Silico Design of Functional DNA Constructs . . . . . . . . . . . . . . . . . . . . . . . 197

Alan Villalobos, Mark Welch, and Jeremy Minshull
16 Using DNAWorks in Designing Oligonucleotides for PCR-Based
Gene Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
David Hoover
17 De Novo Gene Synthesis Design Using TmPrime Software . . . . . . . . . . . . . . . 225
Mo-Huang Li, Marcus Bode, Mo Chao Huang, Wai Chye Cheong,
and Li Shi Lim
18 Design-A-Gene with GeneDesign . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235
Sarah M. Richardson, Steffi Liu, Jef D. Boeke, and Joel S. Bader

PART IV EDUCATION AND SECURITY

19 Leading a Successful iGEM Team . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

Wayne Materi
20 The Build-a-Genome Course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
Eric M. Cooper, Helöise Müller, Srinivasan Chandrasegaran,
Joel S. Bader, and Jef D. Boeke
21 DNA Synthesis Security . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Ali Nouri and Christopher F. Chyba

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Contributors

RINJI AKADA • Department of Applied Molecular Bioscience, Yamaguchi University

Graduate School of Medicine, Ube, Japan
NARAYANA ANNALURU • Department of Environmental Health Sciences,
Johns Hopkins University School of Public Health, Baltimore, MD, USA
JOEL S. BADER • High Throughput Biology Center, Johns Hopkins University School
of Medicine, Baltimore MD, USA; Whiting School of Engineering, Johns Hopkins
University, Baltimore, MD, USA
GWYNEDD A. BENDERS • J. Craig Venter Institute Inc., San Diego, CA, USA
KAJAL BISWAS • Mouse Cancer Genetics Program, Center for Cancer Research,
National Cancer Institute at Frederick, Frederick, MD, USA
MARCUS BODE • Institute of Bioengineering and Nanotechnology,
The Nanos, Singapore
JEF D. BOEKE • Department of Molecular Biology and Genetics, High Throughput
Biology Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA
A. MALCOLM CAMPBELL • Department of Biology, Davidson College, Davidson,
NC, USA; Genome Consortium for Active Teaching, Davidson, NC, USA
JERONIMO CELLO • Department of Molecular Genetics and Microbiology,
Stony Brook University, Stony Brook, NY, USA
KAMONCHAI CHA-AIM • Department of Applied Molecular Bioscience,
Yamaguchi University Graduate School of Medicine, Ube, Japan
SRINIVASAN CHANDRASEGARAN • Department of Environmental Health Sciences,
Johns Hopkins University School of Public Health, Baltimore, MD, USA
SUHWAN CHANG • Mouse Cancer Genetics Program, Center for Cancer Research,
National Cancer Institute at Frederick, Frederick, MD, USA
WAI CHYE CHEONG • Institute of Bioengineering and Nanotechnology, The Nanos,
Singapore
CHRISTOPHER F. CHYBA • Program on Science and Global Security,
Woodrow Wilson School, Princeton University, Princeton, NJ, USA
ERIC M. COOPER • High Throughput Biology Center, Johns Hopkins University
School of Medicine, Baltimore, MD, USA
JESSICA S. DYMOND • High Throughput Biology Center, Johns Hopkins University School of
Medicine, Baltimore, MD, USA
TODD T. ECKDAHL • Department of Biology, Missouri Western State University and Genome
Consortium for Active Teaching., St. Joseph, MO, USA; Genome Consortium for Active
Teaching, Davidson, NC, USA
STEPHEN J. ELLEDGE • Department of Genetics, Howard Hughes Medical Institute,
Harvard Medical School, Boston, MA, USA; Division of Genetics,
Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA
TOMOAKI FUKUNAGA • Department of Applied Molecular Bioscience,
Yamaguchi University Graduate School of Medicine, Ube, Japan

ix
x Contributors

DANIEL G. GIBSON • Department of Synthetic Biology, J. Craig Venter Institute, Inc.,

Rockville, MD, USA
DAVID HOOVER • Scientific Computing Branch, Center for Information Technology,
National Institutes of Health, Bethesda, MD, USA
HISASHI HOSHIDA • Department of Applied Molecular Bioscience, Yamaguchi University
Graduate School of Medicine, Ube, Japan
OLIVIA HO-SHING • Department of Biology, Davidson College, Davidson, NC, USA
MO CHAO HUANG • Institute of Bioengineering and Nanotechnology,
The Nanos, Singapore
KARTHIKEYAN KANDAVELOU • Pondicherry Biotech Private Limited, IT Park,
Pondy Technopolis, Pillaichavady, Puducherry, India
KIN H. LAU • Department of Biology, Davidson College, Davidson, NC, USA
MAMIE Z. LI • Department of Genetics, Howard Hughes Medical Institute, Harvard
Medical School, Boston, MA, USA; Division of Genetics,
Department of Medicine, Brigham and Women’s Hospital, Boston, MA, USA
MO-HUANG LI • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore
LI SHI LIM • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore
GREGORY LINSHIZ • Department of Computer Science and Applied Mathematics,
Weizmann Institute of Science, Rehovot, Israel; Department of Biological Chemistry,
Weizmann Institute of Science, Rehovot, Israel
STEFFI LIU • Department of Biomedical Engineering, Johns Hopkins University, Baltimore,
MD, USA
VIKTORIYA LONDON • Department of Environmental Health Sciences, Johns Hopkins
University School of Public Health, Baltimore, MD, USA
JULIE A. MARCHAND • Virginia Bioinformatics Institute, Virginia Tech, Blacksburg, VA, USA
WAYNE MATERI • Carbonitum Energy Corporation, Edmonton, AB, Canada
JEREMY MINSHULL • DNA2.0, Inc., Menlo Park, CA, USA
STEFFEN MUELLER • Department of Molecular Genetics and Microbiology, Stony Brook
University, Stony Brook, NY, USA
HÉLOÏSE MULLER • Department of Environmental Health Sciences, Johns Hopkins
University School of Public Health, Baltimore, MD, USA
ALI NOURI • Program on Science and Global Security Woodrow Wilson School, Princeton
University, Washington DC, USA
JEAN PECCOUD • Virginia Bioinformatics Institute, Virginia Tech,
Blacksburg, VA, USA
SIVAPRAKASH RAMALINGAM • Department of Environmental Health Sciences, Johns Hopkins
University School of Public Health, Baltimore, MD, USA
SARAH M. RICHARDSON • High Throughput Biology Center, Johns Hopkins University School
of Public Health, Baltimore, MD, USA
JOY WU SCHWERZMANN • Department of Environmental Health Sciences, Johns Hopkins
University School of Public Health, Baltimore, MD, USA
EHUD SHAPIRO • Department of Biological Chemistry, Weizman Institute of Science,
Rehovot, Israel; Department of Computer Science and Applied Mathematics,
Weizman Institute of Science, Rehovot, Israel
SHYAM K. SHARAN • Mouse Cancer Genetics Program, Center for Cancer Research,
National Cancer Institute at Frederick, Frederick, MD, USA
STACEY STAUFFER • Mouse Cancer Genetics Program, Center for Cancer Research, National
Cancer Institute at Frederick, Frederick, MD, USA
 Contributors xi

WILLIAM VERNON • Department of Biology, Missouri Western State University, St. Joseph,
MO, USA
ALAN VILLALOBOS • DNA2.0, Inc., Menlo Park, CA, USA
MARK WELCH • DNA2.0, Inc., Menlo Park, CA, USA
HONGYE YE • Institute of Bioengineering and Nanotechnology, The Nanos, Singapore
TUVAL BEN YEHEZKEL • Department of Biological Chemistry, Weizman Institute of Science,
Rehovot, Israel
 Part I

Assembly of Oligonucleotides in Synthons

 Chapter 1

Building Block Synthesis Using the Polymerase

Chain Assembly Method
Julie A. Marchand and Jean Peccoud

Abstract
De novo gene synthesis allows the creation of custom DNA molecules without the typical constraints of
traditional cloning assembly: scars, restriction site incompatibility, and the quest to find all the desired parts
to name a few. Moreover, with the help of computer-assisted design, the perfect DNA molecule can be
created along with its matching sequence ready to download. The challenge is to build the physical DNA
molecules that have been designed with the software. Although there are several DNA assembly methods,
this section presents and describes a method using the polymerase chain assembly (PCA).

Key words: Gene synthesis, Polymerase chain assembly, Building blocks, DNA fabrication,
Computer-assisted design

1. Introduction

At its core, DNA fabrication relies on the synthesis of DNA oligomers

at base level. The essential feature of DNA fabrication is that no
naturally isolated DNA is used. Although clonal plasmid-based
intermediates might exist during the assembly of a target DNA,
every base originated as a phosphoramidite molecule at the
beginning of the process. Today, all fabrication methods begin
with solid-phase phosphoramidite chemistry to construct single-
stranded DNA molecules that are between 10 and 100 base pairs (bp)
long, which are enzymatically assembled into larger molecules.
This process is commonly referred to as “gene synthesis” and can
be used to synthesize sequences up to 1 kilobase (kb) long. Still
larger target DNA sequences require investigators to assemble
partial products into the desired full-length construct. DNA of
several kb in length can be enzymatically assembled from 1-kb
DNA segments, whereas DNA of megabase length requires in vivo

Jean Peccoud (ed.), Gene Synthesis: Methods and Protocols, Methods in Molecular Biology, vol. 852,
DOI 10.1007/978-1-61779-564-0_1, © Springer Science+Business Media, LLC 2012

3
4 J.A. Marchand and J. Peccoud

Fig. 1. PCA assembly of a DNA construct. A target sequence is shown in the top panel of this figure. The different color
segments represent the oligos that are synthesized to build the construct. The pool of oligos is assembled in equimolar
amounts and allowed to anneal. The annealed oligos are extended in the 3¢ direction until the end of their partner oligo is
reached. The double-stranded DNA is melted and reannealed with extension products and any remaining oligos. Each
extension reaction results in progressively longer products, and full-length products are eventually synthesized. At this
step, the terminal oligos are added to the reaction, and full-length products from the previous reactions are amplified by
PCR and subsequently cloned and sequenced. Figure reproduced with permission from ref. 1.

recombination methods (Fig. 1). The details of DNA fabrication

are therefore not monolithic and are distinct based on the size
of the target DNA (1).
Gene synthesis is the step during which oligonucleotides
(oligos) are combined into DNA fragments of several hundred
bases in length. Numerous protocols have been described and
extensively reviewed, including polymerase chain assembly (PCA),
thermodynamically balanced inside-out synthesis (TBIO) (2), and
 1 Building Block Synthesis Using the Polymerase Chain Assembly Method 5

ligase chain reaction (LCR) (1, 3). Here the PCA method (4) is
used to synthesize 750-bp building blocks from the right arm of
yeast chromosome 6. The right chromosome arm was first subdivided
in 12 segments, each on average 12 kb, and then each segment was
further subdivided in 15 building blocks of 750 bp each. The
building blocks were further dissected into a set of about 12–13
oligonucleotides of about 60 bp in length. The building blocks
were further synthesized from the oligonucleotides using the PCA
method, cloned, and sent for sequencing.

2. Materials

2.1. Software The primers are designed using the software GeneDesign, a web-
based program for the design of synthetic genes (5). It consists of
several modules that automate the tasks associated with the manip-
ulation of synthetic sequences. The source code is from http://
github.com/notadoctor/GeneDesign/.

2.2. Gene Synthesis 1. The primers were purchased from Integrated DNA
and Analysis Technologies, Inc. (IDT, Coralville, IA). They were ordered
wet frozen (in water) in 96-well plates at a concentration of
60 μmol/L and a volume of 83.33 μl/well.
2. HotStarTaq master mix kit.
3. Nuclease-free water (not DEPC-treated).
4. 96-well PCR plates.
5. DNA 12000 kit (catalogue number 5067-1508 Agilent
Technologies, Inc., Wilmington, DE).
6. Agilent Bioanalyzer (Agilent Technologies, Inc., Wilmington,
DE).
7. 96-well block, 1 ml volume/well.

2.3. Cloning 1. TOPO TA cloning® kit for sequencing (catalogue number

of Building Blocks K457501, Life technologies, Carlsbad, CA).
2. TOP10 chemically competent cells (catalogue number
C404003, Life technologies, Carlsbad, CA).
3. Terrific agar plates supplemented with 100 μg/ml carbenicillin.

2.4. Bacterial Culture 1. Luria broth (LB) medium supplemented with 10% glycerol
and 50 μg/ml carbenicillin.
2. SOC medium: complement 1 L of LB medium with 10 ml of
1 M MgSO4 (10 mM final), 10 ml of 1 M MgCl2 (10 mM
final), and 18 ml of 20% dextrose (0.36% w/vol).
3. Air-permeable sealing membrane.
6 J.A. Marchand and J. Peccoud

4. Aluminum seal film.

5. 96-well culture plates.
6. Carbenicillin.

3. Methods

The GeneDesign software (5) allows for breaking large sequences

into several building blocks of about 750 bp, which, in turn, are
further dissected into about 12–13 60-bp oligonucleotides used
for the subsequent synthesis. The building blocks are synthesized
manually here, but this can also be performed with a high-throughput
liquid handling system. To create the DNA template for the build-
ing blocks, this protocol uses the polymerase chain assembly (PCA)
method in which oligonucleotides, present in equimolar quantity,
span both strands of a DNA sequence, anneal through partial over-
lap, and are extended in such a way that each are increased in length
and can be extended further by hybridizing to other oligonucle-
otides or products of subsequent extensions (1). Upon its creation,
the full-length DNA template corresponding to a specific building
block is amplified by polymerase chain reaction (PCR) using the
two outermost oligonucleotides from the PCA step. Following the
completion of the final amplification step, analysis of the PCR
reaction by electrophoresis is used to visualize the presence and
size of the generated building block. This final amplicon is then
cloned in a TOPO®TA cloning vector and transformed. For each
synthesized building block, an average of 12 clones are sent for
sequencing in the form of liquid culture. To avoid template degra-
dation, it is important to clone the amplicons immediately after
the electrophoresis.

3.1. Design of the 1. Using the GeneDesign software (see Subheading 2.1), the
Building Blocks 750-bp building block sequences are entered in the program
using option X (calibrate the Tm, length restriction sites
added). This function will subdivide the sequences in several
primers (usually 12–13 primers, but can be up to 20 for a
longer building block), each with an average length of 60 bp,
and will also deliver a list of primers.
2. Using the primer list, the primers are ordered from commercial
suppliers as described in Subheading 2.2.

3.2. Building Block 1. Using a multichannel pipette, the primers are diluted to 6 μM
Synthesis in nuclease-free water to a final volume of 100 μl in a second
96-well block to produce the working stock. From this 6 μM
primer working stock, a template primer mix (TPM) and outer
primer mix (OPM) are prepared.
 1 Building Block Synthesis Using the Polymerase Chain Assembly Method 7

2. In the TPM, all primers must be present at a concentration

of 300 nM (the primers must all be diluted by 1/20). These
dilutions are prepared in a 0.5-ml reaction tube, and primer
mixes are stored at −20°C when not in use. To prepare the
TPM for a building block that consists of up to 20 primers,
add 10 μl of each primer and if less than 20 primers are used,
add nuclease-free water instead to yield a final volume of
200 μl; mix thoroughly. If more than 20 primers are used, add
10 μl of each primer, but no additional water. The primer con-
centration should be around 250–275 nM.
3. In the OPM, the outer primers must be present at a concentra-
tion of 3 μM (i.e., both primers are diluted by 1/2). Again,
these dilutions are prepared in a 0.5-ml reaction tube in nuclease-
free water, and primer mixes are stored at −20°C when not in
use. To obtain the OPM, add 25 μl of the first and last primer
and mix thoroughly to give a total volume of 50 μl.
4. The building block template synthesis is performed by poly-
merase chain assembly (PCA) in a thermocycler. This reaction
is also called templateless PCR; it has a final volume of 25 μl
and contains the following reagents: 12.5 μl of HotStarTaq
master mix 2× (containing the buffer, the dNTPS, and the Taq
polymerase), 2.5 μl of TPM, and 10 μl of nuclease-free water.
The assembly is performed using the following program: 95°C
for 15 min, 55°C for 30 s, and 72°C for 1 min; then 25 cycles
of 95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; followed
by 72°C for 3 min and 10°C forever (see Note 1).
5. The building block templates are then amplified by PCR using
the OPM to create the finished PCR and thus the building
blocks. This reaction has a final volume of 25 μl and contains
the following reagents: 12.5 μl of HotStarTaq master mix
2× (the buffer, the dNTPS, and the Taq polymerase), 2.5 μl of
templateless PCR reaction from step 4 above (diluted 1:5),
2 μl of OPM, and 10 μl of nuclease-free water. The amplifica-
tion is performed with the following program: 95°C for
15 min, 55°C for 30 s, and 72°C for 1 min; 25 cycles of
95°C for 30 s, 55°C for 30 s, and 72°C for 1 min; followed by
72°C for 3 min and 10°C forever (see Note 1).
6. The building block synthesis is monitored with a microfluidic
electrophoresis using the DNA 12000 kit and the Bioanalyzer
instrument from Agilent according to the manufacturer’s
instructions (Fig. 2). This analysis can also be performed with
an agarose gel.

3.3. Cloning 1. The building blocks are cloned in the TOPO®TA cloning vector
of Building Blocks pCR4® (see Subheading 2.3). All reagents are provided in the
kit. The ligation reaction is composed of 2 μl of the PCR reac-
tion from step 6 above (see Note 2), 1 μl of ultra salt solution,
8 J.A. Marchand and J. Peccoud

Fig. 2. Gel electrophoresis for a selection of synthesized building blocks from segment 15 of the right arm of yeast chromo-
some 6. These building blocks were synthesized using the PCA method, and a microfluidic electrophoresis was performed
to monitor the success of the synthesis. For each building block, positive synthesis is demonstrated by the presence of a
band with a molecular weight that corresponds to the associated building block.

2 μl of ultra pure water, and 1 μl of pCR4® TOPO® vector,

given a final volume of 6 μl. The reagents are added in the
specified order, and the reaction is incubated at room tempera-
ture for 12 min (see Note 3).
2. The cloned building blocks are transformed into chemically
competent bacterial cells E. coli TOP10 (see Note 4). The
entire ligation reaction (6 μl) is added to 50 μl of chemically
competent E. coli TOP10 cells and incubated on ice for 30 min,
before being heat-shocked at 42°C for 30 s and placed back
onto ice for 2 min. Then 400 μl of SOC medium without anti-
biotics is added, and the cells are incubated at 37°C for 1 h.
Finally, the cells are spread on terrific agar plates supplemented
with carbenicillin at 100 μg/ml and incubated at 37°C for
18 h (see Note 6). The vector has a negative selection with
E. coli lethal gene ccdB fused to LacZα fragment. Upon ligation
of an insert, the LacZα-ccdB gene fusion expression is disrupted,
and thus the cell survives. The host must not express the ccdA
gene (see Note 5).

3.4. Culturing of 1. For each construct, 12 colonies are manually picked from the
Clones for Sequencing 18-h agar plates and inoculated in a 96-well culture plate con-
taining 200 μl of LB broth supplemented with 10% glycerol
and 50 μg/ml carbenicillin (see Note 6). The plate is sealed
 1 Building Block Synthesis Using the Polymerase Chain Assembly Method 9

with an air-permeable membrane and incubated without

agitation at 37°C for 18 h.
2. The 18-h plate is then used to inoculate two replicate 96-well
culture plates. Using a multichannel pipette, 10 μl of the 18-h
culture plate is transferred to each of the 96-well plates con-
taining 190 μl of LB broth supplemented with 10% glycerol
and 50 μg/ml carbenicillin. The plates are sealed with an air-
permeable membrane and incubated without agitation at 37°C
for exactly 12 h. The 18-h plate is sealed with aluminum seal
and stored at −80°C.
3. Upon completion of the incubation time, the two 12-h plates
are sealed with aluminum seal and stored at −80°C. One of the
12-h plates will be sent to an external company for sequencing,
and the other is kept.
4. After the sequencing results are analyzed, the selected perfect
clones are picked from their respective culture plates and grown
in tubes containing 3 ml of LB broth supplemented with 10%
glycerol and 100 μg/ml carbenicillin and incubated in an incu-
bator shaker at 37°C and 250 RPM for exactly 12 h. The culture
is then frozen as a glycerol stock in a cryogenic vial. The 96-well
plates containing the incorrect clones are discarded.

4. Notes

1. The annealing temperature for PCA or finishing PCR reaction

must be adjusted according to the Tm of the primers.
2. The volume of PCR reactions used for the ligation can range
from 0.5 μl to 4 μl depending on the yield of the amplicons.
3. The building block can also be cloned by traditional ligation
provided that restriction sites are added on the outermost
primers, or by cloning systems such as Gateway® that has
recombination sites added to the outmost primers. If a uracil-
specific excision reagent (USER) fusion system is used, it is
important to note that wild-type archaeal DNA polymerases
are inhibited by the deoxyuracil.
4. The selection of the bacterial strain should be made according
to both the strain genotype and the type of insert. For the
expression of yeast parts, the E. coli TOP10 cells appear more
suitable, and we have observed increased number of colonies
and good growth with this strain.
5. Despite the presence of the ccdB lethal gene, we have observed
some negative colonies that do not appear to be satellite colonies.
Random screening or sending more clones for sequencing
might be advisable.
10 J.A. Marchand and J. Peccoud

6. This growth medium is recommended by Beckman Coulter

Genomics where our group receives the sequencing service.
The selective antibiotic is based on the resistance encoded by
the vector of choice.

References

1. Czar MJ, Anderson JC, Bader JS and 4. Dymond J, Scheifele L, Richardson S, Lee P,
Peccoud J. (2009) Gene synthesis demystified. Chandrasegaran S, Bader J and Boeke JD
Trends Biotechnol 27:63–72. (2009) Teaching Synthetic Biology, Bioinfor-
2. Xiong AS, Peng RH, Zhuang J, Gao F, Li Y, matics, and Engineering to Undergraduates:
Cheng Z M and Yao, QH (2008) Chemical gene The Interdisciplinary Build-a-Genome Course.
synthesis: strategies, softwares, error corrections, Genetics 18:13–21.
and applications. FEMS Microbiol 32:522–540. 5. Richardson SM, Wheelan SJ, Yarrington, RM
3. Cello J, Paul AV and Wimmer E (2002) Chemical and Boeke, JD (2006) GeneDesign: rapid, auto-
Synthesis of Poliovirus cDNA: Generation of mated design of multikilobase synthetic genes.
Infectious Virus in the Absence of Natural Genome Res 16:550–6.
Template. Science 297:1016–1018.
 Chapter 2

Oligonucleotide Assembly in Yeast to Produce

Synthetic DNA Fragments
Daniel G. Gibson

Abstract
The yeast Saccharomyces cerevisiae can take up and assemble at least 38 overlapping single-stranded
oligonucleotides and a linear double-stranded vector in one transformation event. These oligonucleotides
can overlap by as few as 20 bp and can be as long as 200 nucleotides in length to produce kilobase-sized
synthetic DNA molecules. A protocol for designing the oligonucleotides to be assembled, transforming
them into yeast, and confirming their assembly is described here. This straightforward scheme for assem-
bling chemically synthesized oligonucleotides can be a useful tool for building synthetic DNA molecules.

Key words: In vivo DNA assembly, Yeast transformation, Gene synthesis, Oligonucleotides, Synthetic
biology

1. Introduction

Chemically synthesized oligonucleotides (oligos) are often joined

into larger DNA fragments containing full-length genes. This was
first demonstrated in 1970 when Khorana and colleagues synthe-
sized the 77-nucleotide gene encoding a yeast alanine transfer RNA
from 17 overlapping oligonucleotides (1). Since then, chemical oli-
gonucleotide synthesis has improved tremendously (2), and a num-
ber of in vitro enzymatic strategies are available for the assembly of
oligos into larger constructs (3–5). It is now possible to produce
genes, biosynthetic pathways, and even entire chromosomes from
chemically synthesized DNA (6, 7). Because absolute control can
be exerted over the sequence of chemically derived DNA mole-
cules, genetic components can be exhaustively optimized.
The capacity of the yeast Saccharomyces cerevisiae to take up
and recombine DNA fragments has made it a model eukaryote for
studying numerous cellular processes. This is mainly because DNA
sequences can be genetically altered by transforming yeast with

Jean Peccoud (ed.), Gene Synthesis: Methods and Protocols, Methods in Molecular Biology, vol. 852,
DOI 10.1007/978-1-61779-564-0_2, © Springer Science+Business Media, LLC 2012

11
12 D.G. Gibson

either double-stranded (ds) DNA fragments (8) or single-stranded

(ss) oligos (9). In addition, homologous recombination in yeast can
be used to build DNA fragments from overlapping constituent parts.
This was first demonstrated when a plasmid was constructed from
two dsDNA fragments containing homologous ends (10). Two
nonhomologous dsDNA fragments can also be bridged by single-
stranded oligonucleotides that join the ends of the two fragments
(11). Previously, we showed that six overlapping dsDNA fragments
could be assembled by yeast into an entire Mycoplasma genitalium
genome (6). Subsequently, this process was improved, and 25 over-
lapping fragments, between 17 kb and 35 kb in length, were assem-
bled at once into this genome (12). More recently, we reported on
the synthesis of a 1.08-Mbp Mycoplasma mycoides genome, which
was used to produce a cell controlled only by this synthetic genome
(7). Using yeast recombination, the synthetic M. mycoides genome
was assembled in three stages from 1,078 overlapping 1,080-bp
DNA fragments that were each chemically synthesized.
To exclusively use yeast in the production of whole genomes
and large constructs of any reasonable sequence, what remained
was the demonstration of the assembly of chemically synthesized
oligonucleotides into appropriate dsDNA molecules, which we
reported in 2009 (13). There we showed that yeast could take up
and assemble at least 38 overlapping single-stranded oligonucle-
otides and a linear double-stranded vector in one transformation
event to produce ~1.2-kb dsDNA fragments. These oligonucle-
otides can overlap by as few as 20 bp and can be as long as 200
nucleotides in length. A protocol for synthesizing kilobase-sized
DNA fragments in yeast from a series of overlapping oligos is
described here.

2. Materials

2.1. Design 1. Yeast/E. coli shuttle vector [e.g., pRS313 (ATCC 77142),
and Preparation pRS314 (ATCC 77143), pRS315 (ATCC 77144), and pRS316
of the Oligonucleotides (ATCC 77145)].
and Assembly Vector 2. Primers for assembly vector amplification.
3. Overlapping synthetic oligonucleotides to be assembled.
4. High-fidelity polymerase chain reaction (PCR) amplification
kit (e.g., Phusion® polymerase (New England BioLabs®,
Inc. [NEB])).
5. Gel extraction kit (e.g., QIAquick Gel Extraction Kit, Qiagen).
6. Tris–EDTA buffer pH 8.0 (TE buffer).
7. DNA analysis software (e.g., Vector NTI® [Invitrogen],
Clone Manager [Sci-Ed], and CLC Genomics Workbench
[CLC bio]).
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*** START OF THE PROJECT GUTENBERG EBOOK DEVOTIONAL

POETRY FOR THE CHILDREN. SECOND PART ***
DEVOTIONAL POETRY
FOR THE

CHILDREN.

SECOND PART.

“Make us beautiful within,

By Thy Spirit’s holy light;
Guard us when our faith burns dim,
Father of all love and might.”

PHILADELPHIA:
Published by the Book Association of Friends.
1870.
 Electrotyped and Printed for the Association,
By THOMAS W. STUCKEY,
403 North Sixth street, above Callowhill, Philadelphia.
 INDEX.

PAGE
The Life-Clock, 5
God is Love, 6
Time, 7
Thanksgiving, 7
“Thou, God, seest Me,” 8
The Beautiful Works of God, 9
Spiritual Blessings, 10
The Dove’s Visit, 10
Teach Us to Pray, 12
Deeds of Kindness, 12
An Evening Song, 14
Be Kind to The Poor, 15
The Lesson of The Leaves, 16
The Spring-Bird’s Lesson, 17
The Orphan’s Hymn, 18
Morning, 18
Evening, 19
A Moment Too Late, 20
A Little Sonnet about Little Things, 21
Examination, 22
God is in His holy Temple, 23
Morning Glories, 24
How Beautiful the Setting Sun, 25
Summer Time, 26
Like Jesus, 27
I Have a Home, 27
God, 28
The Bird’s Nest, 29
The Lark, 30
Effort, 30
The Sea Shell, 31
God is Good, 32
Despise not Simple Things, 32
The Violet, 33
Child’s Talent, 34
The Stars are Coming, 35
The Flowers, 36
Little by Little, 37
Never, My Child, Forget to Pray, 38
The Child’s Prayer, 38
A Childlike Spirit, 39
Live for Something, 41
The Beautiful, 42
Don’t Kill the Birds, 43
Little Acts of Kindness, 44
The Blessings, 46
When Father Comes Home, 47
Harvest-Field of Time, 48
Prayer, 49
Reflections, 49
What is Heaven? 50
The Child’s Monitor, 51
Give Us our Daily Bread, 52
True Rest, 54
One by One, 56
God Seen in His Works, 57
The Little Sunbeam, 58
Compassion, 59
I Will be Good to-day, 59
I’ll Do what I Can, 60
Time to Arise, 61
Divine Guidance, 62
Industry, 62
“Prayer is the Soul’s sincere Desire,” 63
Angry Words, 63
The Request, 64
DEVOTIONAL POETRY

FOR THE

CHILDREN.
 THE LIFE-CLOCK.
There is a little mystic clock,
No human eye hath seen,
That beateth on,—and beateth on,—
From morning until e’en.

And when the soul is wrapped in sleep,

All silent and alone,
It ticks and ticks the livelong night,
And never runneth down.

Oh! wondrous is that work of art,

Which knells the passing hour;
But art ne’er formed, nor mind conceived,
The life-clock’s magic power.

Not set in gold, nor decked with gems,

By wealth and pride possessed;
But rich or poor, or high or low,
Each bears it in his breast.

Such is the clock that measures life,—

Of flesh and spirit blended,—
And thus ’t will run within the breast,
Till that strange life is ended.
 GOD IS LOVE.
Lo! the heavens are breaking,
Pure and bright above;
Light and life awaking,
Murmur, “God is love.”

Music now is ringing,

Through the leafy grove,
Feathered songsters, singing,
Warble, “God is love.”

Wake, my heart, and springing,

Spread thy wings above;
Soaring still, and singing,—
Singing, “God is love.”
 TIME.
A minute,—how soon it is flown!
And yet, how important it is!
God calls every moment His own,—
For all our existence is His:
And tho’ we may waste many moments each day,
He notices each that we squander away.

We should not a minute despise,

Although it so quickly is o’er;
We know that it rapidly flies,
And therefore should prize it the more.
Another, indeed, may appear in its stead;
But that precious minute, for ever, is fled.

’Tis easy to squander our years

In idleness, folly, and strife;
But, oh! no repentance nor tears
Can bring back one moment of life.
Then wisely improve all the time as it goes,
And life will be happy, and peaceful the close.
 THANKSGIVING.
There’s not a leaf within the bower,—
There’s not a bird upon the tree,—
There’s not a dewdrop on the flower,—
But bears the impress, Lord, of Thee.

Thy power the varied leaf designed,

And gave the bird its thrilling tone;
Thy hand the dewdrops’ tints combined,
Till like a diamond’s blaze they shone.

Yes, dewdrops, leaves and buds, and all,—

The smallest, like the greatest things,—
The sea’s vast space, the earth’s wide ball,
Alike proclaim Thee, King of kings!

But man alone, to bounteous Heaven,

Thanksgiving’s conscious strains can raise:
To favored man, alone, ’tis given,
To join the angelic choir in praise.
 “THOU, GOD, SEEST ME.”
Thine eye is on me always,
Thou knowest the way I take;
Thou seest me when I’m sleeping,
Thou seest me when I wake.

Thine arm is round about me,

Thy hand is underneath;
Thy love will still preserve me,
If I Thy laws do keep.

Thou art my present helper,—

Be Thou my daily guide;
Then I’ll be safe for ever,
Whatever may betide.

Oh! help me, dearest Father,

To walk in wisdom’s way,
That I, Thy loving child, may be
Through every future day,
And, by my loving actions, prove
That He who guardeth me is Love.
 THE BEAUTIFUL WORKS OF GOD.
All things bright and beautiful,
All creatures great and small,
All things wise and wonderful,—
The Lord God made them all.

Each little flower that opens,

Each little bird that sings,
He made their glowing colors,
He made their shining wings.

The tall trees in the green wood,

The meadows where we play,
The rushes, by the water,
We gather every day,—

He gave us eyes to see them,

And lips, that we may tell
How great is God Almighty,
Who doeth all things well.
 SPIRITUAL BLESSINGS.
Almighty Father! Thou hast many blessings
In store for every loving child of Thine;
For this I pray,—Let me, Thy grace possessing,
Seek to be guided by Thy will divine.

Not for earth’s treasures,—for her joys the dearest,—

Would I my supplications raise to Thee;
Not for the hopes that to my heart are nearest,
But only that I give that heart to Thee.

I pray that Thou wouldst guide and guard me ever;

Cleanse, by Thy power, from every stain of sin;
I will Thy blessing ask on each endeavor,
And thus Thy promised peace my soul shall win.
 THE DOVE’S VISIT.
I knew a little, sickly child,
The long, long summer’s day,
When all the world was green and bright,
Alone in bed to lay;
There used to come a little dove
Before his window small,
And sing to him with her sweet voice,
Out of the fir-tree tall.

And when the sick child better grew,

And he could creep along,
Close to that window he would come,
And listen to her song.
He was so gentle in his speech,
And quiet at his play,
He would not, for the world, have made,
That sweet bird fly away.

There is a Holy Dove that sings

To every listening child,—
That whispers to his little heart
A song more sweet and mild.
It is the Spirit of our God
That speaks to him within;
That leads him on to all things good,
And holds him back from sin.

And he must hear that “still, small voice,”

Nor tempt it to depart,—
The Spirit, great and wonderful,
 That whispers in his heart.
He must be pure, and good, and true;
Must strive, and watch, and pray;
For unresisted sin, at last,
May drive that Dove away.
 TEACH US TO PRAY.
Teach us to pray
Oh, Father! we look up to Thee,
And this our one request shall be,
Teach us to pray.

Teach us to pray.
A form of words will not suffice,—
The heart must bring its sacrifice:
Teach us to pray.

Teach us to pray.
To whom shall we, Thy children, turn?
Teach Thou the lesson we would learn:
Teach us to pray.

Teach us to pray.
To Thee, alone, our hearts look up:
Prayer is our only door of hope;
Teach us to pray.
 DEEDS OF KINDNESS.
Suppose the little cowslip
Should hang its tiny cup,
And say, “I’m such a little flower,
I’d better not grow up.”
How many a weary traveler
Would miss the fragrant smell?
How many a little child would grieve
To miss it from the dell!

Suppose the glistening dew-drop,

Upon the grass, should say,
“What can a little dew-drop do?
I’d better roll away.”
The blade on which it rested,
Before the day was done,
Without a drop to moisten it,
Would wither in the sun.

Suppose the little breezes

Upon a summer’s day,
Should think themselves too small to cool
The traveler on his way:
Who would not miss the smallest
And softest ones that blow,
And think they made a great mistake
If they were talking so?

How many deeds of kindness

A little child may do,
Although it has so little strength,
 And little wisdom, too.
It wants a loving spirit,
Much more than strength, to prove,
How many things a child may do
For others by his love.
 AN EVENING SONG.
How radiant the evening skies!
Broad wing of blue in heaven unfurled,
God watching with unwearied eyes
The welfare of a sleeping world.

He rolls the sun to its decline,

And speeds it on to realms afar,
To let the modest glowworm shine,
And men behold the evening star.

He lights the wild flower in the wood,

He rocks the sparrow in her nest,
He guides the angels on their road,
That come to guard us while we rest

When blows the bee his tiny horn,

To wake the sisterhood of flowers,
He kindles with His smile the morn,
To bless with light the winged hours.

O God! look down with loving eyes

Upon Thy children slumbering here,
Beneath this tent of starry skies,
For heaven is nigh, and Thou art near.
 BE KIND TO THE POOR.
Turn not from him, who asks of thee
A portion of thy store;
Poor though in earthly goods thou be,
Thou yet canst give,—what’s more,

The balm of comfort thou canst pour

Into his grieving mind,
Who oft is turned from wealth’s proud door,
With many a word unkind.

Does any from the false world find

Naught but reproach and scorn?
Does any, stung by words unkind,
Wish that he ne’er was born?

Do thou raise up his drooping heart,

Restore his wounded mind;
Though naught of wealth thou canst impart,
Yet still thou mayest be kind.

And oft again thy words shall wing

Backward their course to thee,
And in thy breast will prove a spring
Of pure felicity.
 THE LESSON OF THE LEAVES.
How do the leaves grow,
In spring, upon their stems?
Oh! the sap swells up with a drop for all,
And that is life to them.

What do the leaves do

Through the long summer hours,
They make a home for the wandering birds,
And shelter the wild flowers.

How do the leaves fade

Beneath the autumn blast?
Oh! they fairer grow before they die,
Their brightest is their last.

We, too, are like leaves,

O children! weak and small;
God knows each leaf of the forest shade:
He knows us, each and all.

Never a leaf falls

Until its part is done;
God gives us grace, like sap, and then
Some work to every one.

We, too, must grow old,

Beneath the autumn sky;
But lovelier and brighter our lives may grow,
Like leaves before they die.
 Brighter with kind deeds,
With love to others given;
Till the leaf falls off from the autumn tree,
And the spirit is in heaven.
 THE SPRING BIRD’S LESSON.
Thou’rt up betimes, my little bird,
And out this morning early,
For still the tender bud is closed,
And still the grass is pearly.

Why rise so soon, thou little bird,

Thy soft, warm nest forsaking?
To brave the dull, cold morning sky,
While day is scarcely breaking?

Ah! thou art wise, thou little bird,

For fast the hours are flying;
And this young day, but dawning now,
Will soon, alas! be dying.

I’ll learn of thee, thou little bird,

And slothful habits scorning,
No longer sleep youth’s dawn away,
Nor waste life’s precious morning.
 THE ORPHAN’S HYMN.
Father,—an orphan’s prayer receive,
And listen to my plaintive cry:
Thou only canst my wants relieve,
Who art my Father in the sky.

I have no father here below,

No mother kind to wipe my tears,—
These tender names I never know,
To soothe my grief and quell my fears.

But Thou wilt be my parent,—nigh

In every hour of deep distress,
And listen to an orphan’s sigh,
And soothe the anguish of my breast.

For Thou hast promised all I need,

More than a father’s, mother’s care:
Thou wilt the hungry orphan feed,
And always listen to my prayer.
 MORNING.
Dear Lord, another day has come,
And through the hours of night,
In a good bed and quiet home
I’ve slept till morning light.

Then let me give Thee thanks and praise,

For Thou art very good;
Oh, teach my little heart to raise
The prayer that children should.

Keep me this day from faults and sin,

And make me good and mild;
Thy Holy Spirit place within,
Grant grace unto a child.

Help me obey my parents dear,

For they are very kind;
And when the hour of rest draws near,
Another prayer I’ll find.
 EVENING.
The day is gone,—the silent night
Invites me to my peaceful bed;
But, Lord, I know that it is right
To thank Thee, ere I rest my head.

For my good meals and pleasant hours,

That I have had this present day,
Let me exert my infant powers
To praise Thee, nor forget to pray.

Thou art most good. I can’t tell all

That Thou hast ever done for me;
My Shepherd, now on Thee I call,
From dangers still preserve me free.

If I’ve been naughty on this day,

Oh! make me sorry for my fault;
Do Thou forgive, and teach the way
To follow Jesus as I ought.

And now I’ll lay me down to rest,

Myself,—my friends,—all safely keep;
May Thy great name be ever blest,
Both when we wake, and when we sleep.
 A MOMENT TOO LATE!
A moment too late, my beautiful bird,—
A moment too late are you now,
The wind has your soft, downy nest disturbed,—
The nest that you hung on the bough.
A moment too late,—that string in your bill
Would have fastened it firmly and strong;
But see, there it goes rolling over the hill!
Oh! you tarried a moment too long.

A moment too late,—too late, busy bee,

The honey has dropped from the flower;
No use to creep under the petals to see,—
It stood ready to drop for an hour.
A moment too late,—had you sped on your wing,
The honey would not have been gone;
But see what a very,—a very sad thing,
’Tis to tarry a moment too long.
 A LITTLE SONNET ABOUT LITTLE THINGS.
The little, smoky vapors
Produce the drops of rain;
These little drops commingle,
And form the boundless main.

Then, drops compose the fountains;

And little grains of sand
Compose the mighty mountains,
That high above us stand.

The little atoms, it is said,

Compose the solid earth;
Such truths will show, if rightly read,
What little things are worth.

For, as the sea of drops is made,

So it is Heaven’s plan,
That atoms should compose the globe,
And actions mark the man.

The little seconds soon pass by,

And leave our time the less;
And on these moments, as they fly,
Hang woe or happiness.

For, as the present hour is spent,

So must the future be;
Each action lives, in its effect,
Through all eternity.
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