Characterizing seamless ligation cloning extract for synthetic biological applications

Accepted Manuscript Characterizing Seamless Ligation Cloning Extract for Synthetic Biological Applications Katrin Messerschmidt, Lena Hochrein, Daniel Dehm, Karina Schulz, Bernd MuellerRoeber PII:

S0003-2697(16)30114-2

DOI:

10.1016/j.ab.2016.05.029

Reference:

YABIO 12402

To appear in:

Analytical Biochemistry

Received Date: 8 March 2016 Revised Date:

30 May 2016

Accepted Date: 31 May 2016

Please cite this article as: K. Messerschmidt, L. Hochrein, D. Dehm, K. Schulz, B. Mueller-Roeber, Characterizing Seamless Ligation Cloning Extract for Synthetic Biological Applications, Analytical Biochemistry (2016), doi: 10.1016/j.ab.2016.05.029. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Independent of GC content

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Modification of vector

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ACCEPTED MANUSCRIPT 1 2 3

Characterizing Seamless Ligation Cloning Extract for Synthetic Biological Applications

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Potsdam, Germany

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Katrin Messerschmidt1,*, Lena Hochrein1, Daniel Dehm1, Karina Schulz1, Bernd Mueller-Roeber2,3 University of Potsdam, Cell2Fab Research Unit, Karl-Liebknecht-Str. 24-25, 14476 Potsdam, Germany

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University of Potsdam, Department of Molecular Biology, Karl-Liebknecht-Str. 24-25, 14476

Center of Plant Systems Biology and Biotechnology (CPSBB), Ruski Blvd. 139, Plovdiv 4000, Bulgaria

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Corresponding author: Katrin Messerschmidt, *Email: [email protected]

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10 Abstract

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Synthetic biology aims at designing and engineering organisms. The engineering process typically

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requires the establishment of suitable DNA constructs generated through fusion of multiple protein

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coding and regulatory sequences. Conventional cloning techniques, including those involving

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restriction enzymes and ligases, are often of limited scope, in particular when many DNA fragments

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must be joined or scar-free fusions are mandatory. Overlap based-cloning methods have the

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potential to overcome such limitations. One such method uses Seamless Ligation Cloning Extract

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(SLiCE) prepared from Escherichia coli cells for straightforward and efficient in vitro fusion of DNA

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fragments. Here, we systematically characterized extracts prepared from the unmodified E. coli strain

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DH10B for SLiCE-mediated cloning and determined DNA sequence-associated parameters that affect

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cloning efficiency. Our data revealed the virtual absence of length restrictions for vector backbone

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(up to 13.5 kbp) and insert (90 bp to 1.6 kbp). Furthermore, differences in GC content in homology

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regions are easily tolerated and the deletion of unwanted vector sequences concomitant with

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targeted fragment insertion is straightforward. Thus, SLiCE represents a highly versatile DNA fusion

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method suitable for cloning projects in virtually all molecular and synthetic biology projects.

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Running title: SLiCE properties

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Keywords: SLiCE, seamless ligation cloning, homologous recombination, synthetic biology

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1 Introduction

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Synthetic biology aims at designing and engineering novel biological systems, as well as at

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redesigning naturally existing organisms1-3, for example by transferring a biosynthetic pathway for a

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secondary metabolite from its original organism (e.g. a plant) into a more docile heterologous host,

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where the compound of interest can be produced more easily in large quantities2-4. In such exercises,

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the cloning of a large number of DNA pieces is often needed, not only for the insertion of different

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protein coding sequences but also of regulatory sequences such as promoters, terminators, 1

ACCEPTED MANUSCRIPT insulators and others. Conventional cloning techniques use restriction enzymes (RE) to generate

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compatible ends for the ligation of insert and vector5. Consequently, the RE recognition site has to be

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present in the multiple cloning site of the vector but should typically not occur within the insert

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sequence. Considering the large number of genes and regulatory sequences that need to be

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combined for creating new biosynthetic pathways or even synthetic organisms with new features,

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RE-based cloning methods are difficult to employ for such purposes. Furthermore, scar sequences

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typically remain in the construct that might affect the activity of the assembled DNA pieces6. To

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overcome some of the drawbacks inherent to RE-mediated cloning, several new cloning techniques

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were established in recent years that facilitate the assembly of multiple DNA fragments7. Examples

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include BioBricks8, BglBricks9, and GoldenGate cloning10. However, also these methods rely on REs,

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which might leave scar sequences in the constructs produced. Finally, methods employing homology

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regions were established. These methods are essentially independent of vector and insert sequences

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and they do not leave behind scar sequences. Well known technologies of this category are Gibson

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assembly11 and transformation-assisted recombination (TAR) in yeast (Saccharomyces cerevisiae)

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host cells12. In addition, cell extracts prepared from Escherichia coli can be used for seamless ligation

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cloning (Seamless Ligation Cloning Extract = SLiCE13, 14). Although the three methods rely on different

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reaction principles, requirements to vectors and inserts are virtually identical: a linearized vector

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backbone is obtained by cutting with a RE or by PCR amplification, and DNA fragments to be inserted

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are PCR amplified and in this way equipped at both ends with sequences identical to the ends of the

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linearized vector. As Gibson assembly employs three different enzymes, namely T5 exonuclease,

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Phusion polymerase, and Taq ligase, cloning with this particular method is rather cost intensive. A

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drawback of the cheaper TAR-based cloning is the slow growth of yeast cells, which makes the

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method relatively time consuming. Compared to Gibson assembly and TAR, cloning with SLiCE is a

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fast and low-cost alternative. Using SLiCE, DNA fragments are assembled in vitro using bacterial (E.

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coli) extracts. Furthermore, cloning capabilities and efficiencies of the bacterial extract can be

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improved by introducing the lambda prophage Red recombination system into E. coli DH10B (new

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strain named PPY)13. Other studies showed that a broad range of E. coli strains can be used to

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generate seamless ligation cloning extracts15. Furthermore, experimental evidence indicates that the

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method employed for lysate preparation has a significant impact on cloning efficiency16. Here, we

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provide a systematic characterization of SLiCE-based cloning with extracts prepared from the

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unmodified, widely used lab strain E. coli DH10B.

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2 Materials and methods

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2.1 Bacterial strains

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Escherichia coli DH10B (F- endA1 recA1 galE15 galK16 nupG rpsL ΔlacX74 Φ80lacZΔM15 araD139 2

ACCEPTED MANUSCRIPT Δ(ara,leu)7697 mcrA Δ(mrr-hsdRMS-mcrBC λ-), Life Technologies, Carlsbad, USA) was used to

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generate seamless ligation cloning extract (SLiCE). SLiCE-generated constructs were transformed

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either into competent E. coli DH5-alpha cells (fhuA2 lac(del)U169 phoA glnV44 Φ80' lacZ(del)M15

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gyrA96 recA1 relA1 endA1 thi-1 hsdR17) prepared in our lab or into NEB 5-alpha cells purchased from

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New England Biolabs (Frankfurt am Main, Germany).

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2.2 Generation and transformation of competent E. coli cells

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Chemically competent E. coli cells were generated as follows. Cells from a cryostock were plated on

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LB agar plates (yeast extract 5 g/l, tryptone 10 g/l, NaCl 10g/l, and 1.5% [w/v] bacto-agar) and

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incubated overnight at 37°C. A single colony was inoculated into 5 ml LB medium (yeast extract 5 g/l,

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tryptone 10 g/l, NaCl 10g/l) and again incubated overnight at 230 rpm and 37°C followed by

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inoculation of 1 ml of overnight culture into 50 ml LB medium and further incubation at 230 rpm and

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37°C until a cell density of OD600 nm > 0.5 was reached. Cultured cells were cooled on ice for 15 min

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and afterwards centrifuged at 5,000 x g for 5 min at 4°C. Supernatant was discarded and pellet

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resuspended in 25 ml of ice-cold, sterile 100 mM CaCl2 solution. After incubation on ice for 15 min

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cells were centrifuged at 5,000 x g for 5 min at 4°C. Again, the supernatant was discarded and the

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pellet was resuspended in 4 ml ice-cold, sterile 100 mM CaCl2 solution with 15 % [v/v] glycerol,

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aliquoted at 50 µl, frozen in liquid nitrogen, and stored at -80°C. Transformation efficiency of

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competent E. coli cells was tested using a standard protocol and pUC19 DNA (New England Biolabs).

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For transformation of chemically competent E. coli cells, 2 - 4 µl of SLiCE cloning reaction were added

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to 25 - 50 µl competent cells (thawed on ice for 10 min), incubated for 10 min on ice, followed by a

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1 min heat shock at 42°C and incubation on ice for 3 min. Afterwards, 450 µl SOC medium (2% [w/v]

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vegetable peptone, 0.5% [w/v] yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4,

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20 mM glucose) was added and cells were incubated at 37°C and 800 rpm for 1 h before spreading

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150 - 500 µl of the cell suspension onto LB agar plates with corresponding antibiotic (100 µg/ml

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carbenicillin or 30 µg/ml kanamycin). Plates were incubated overnight at 37°C.

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2.3 Preparation of seamless ligation cloning extract

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Preparation of seamless ligation cloning extract (SLiCE) was done as described elsewhere13, with the

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following modifications: cells were inoculated into 20 ml liquid 2 x YT medium (16 g/l tryptone, 10 g/l

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yeast extract, 5 g/l NaCl, pH 7.0) in a 100-ml baffled Erlenmeyer flask and grown overnight at 37°C

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and 200 rpm. The main culture was started on the following morning by transfer of 2 ml of the pre-

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culture into 100 ml 2 x YT medium in a 500-ml baffled Erlenmeyer flask and incubation at 37°C and

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230 rpm until the culture reached OD600nm = 4.5. Cells were pelleted by centrifugation at 5,000 x g for

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20 min at 4°C. Pellets were washed with 50 ml ice-cold ddH2O and pelleted again by centrifugation at 3

ACCEPTED MANUSCRIPT 5,000 x g for 20 min at 4°C. Pellets (0.25 g each) were resuspended in 300 μl CelLytic B Cell Lysis

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Reagent (Sigma-Aldrich, St. Louis, USA) and incubated at room temperature for 10 min for lysis.

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Afterwards, cell lysates were centrifuged at 20,000 x g for 2 min at room temperature to pellet

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insoluble cell debris. Cell extract (supernatant) was mixed with an equal volume of 100% [v/v]

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glycerol and subjected to freezing and storage in three different ways to study the effect on cloning

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efficiency: (i) Extracts were aliquoted at 20 μl in 0.5-ml plastic tubes and immediately transferred

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to -80°C. (ii) Extracts were directly pipetted into liquid nitrogen. Resulting globules of 10 µl frozen

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extract were transferred into 0.5-ml plastic tubes and stored at -80°C. (iii) Extracts were aliquoted

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into 0.2-ml thin-walled plastic tubes, immediately frozen in liquid nitrogen, and afterwards stored at -

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20°C or -80°C. Additionally, these extracts were subjected to repeated freezing and thawing at their

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respective storage temperature. To this end, extracts were thawed on ice and incubated on ice for 10

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min before freezing at the specified temperature again.

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Furthermore, we tried to optimize cell lysis by freezing the cell pellet overnight at -80°C before

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adding CelLytic B Cell Lysis Reagent.

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2.4 SLiCE cloning reactions for systematic characterization

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In each experiment performed to systematically characterize SLiCE cloning reactions one insert of

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linear, double-stranded DNA was mixed with a linearized and dephosphorylated vector backbone for

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homologous recombination. SLiCE reactions were performed as described elsewhere in 10-µl

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reaction volumes13. One µl SLiCE and 1 µl SLiCE buffer (500 mM Tris-HCl, pH 7.5, with 100 mM MgCl2,

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10 mM ATP, and 10 mM DTT) were added to a DNA mixture containing 50 ng linearized vector

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backbone and a suitable amount of insert (molar ratio 1: 10), filled up to 10 µl with sterile water and

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incubated at 37°C for 1 h. Afterwards, 2 - 5 µl of SLiCE reaction were transformed into chemically

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competent E. coli cells (transformation efficiency 1 x 106 -1 x 109 cfu/μg pUC19 DNA), as described

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above.

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2.4.1 Generation of vector backbone

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Vector pYES2/CT (Life Technologies) was used as vector backbone either, unmodified (5,963 bp) or

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modified (see below). The unmodified vector was linearized with either BamHI or HindIII, or both,

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dephosphorylated and purified before use in SLiCE experiments. To obtain the modified vector,

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pYES2/CT was first digested with PmeI and NaeI to remove the GAL1 promoter and multiple cloning

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site (pYes_bkb, 5,143 bp). In a second step, the coding region of the Arabidopsis thaliana protein

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ORE1 (AGI code AT5G39610) was introduced as a 1,389-bp spacer, harboring a single BamHI

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restriction site at position 1,060. The resulting vector (pKM068, 6,532 bp) was linearized by digestion

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with BamHI, dephosphorylated and purified before use in SLiCE experiments. 4

ACCEPTED MANUSCRIPT 142 2.4.2 Generation of inserts

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Two different inserts were used. The insert for the experiment on vector backbone linearization

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encoded His-tagged viral coat protein of hamster polyoma virus17 (VP1, 1,245 bp). The coding region

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was amplified via PCR from vector pKP317 using gene-specific primers (Supplementary Table 1) and

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Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). The PCR reaction components

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were: 50 μl total volume with 1 U polymerase, 10 μl 5x Phusion HF buffer, 200 µM dNTPs, 1 ng of

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plasmid DNA template, and 15 pmol of each primer. The PCR cycling parameters were 98°C 30 s,

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(98°C 10 s, 58°C 30 s, 72°C 1 min) × 30 cycles, 72°C 5 min, and 8°C infinite. The insert used in all other

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reactions for the systematic characterization of SLiCE reactions was the kanamycin resistance gene

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including promoter and terminator (1,016 bp = selectable insert, KanaR), amplified from vector

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pHis2.1 (Clontech Laboratories, Mountain View, USA) using primers specific for the 5’ end of the

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promoter and the 3’ end of the terminator (Supplementary Table 1). The PCR reaction components

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were: 10 μl total volume with 5 µl Maxima Hot Start Green PCR Master Mix (2 x, Thermo Fisher

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Scientific), 0.2 ng of plasmid DNA template, and 3 pmol of each primer. The PCR cycling parameters

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were 95°C 4 min, (95°C 30 s, 64°C 30 s, 72°C 90 s) × 30 cycles, 72°C 5 min, and 8°C infinite.

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158 2.4.3 Primers for insert amplification

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Several primers were used to characterize the various aspects of SLiCE-mediated cloning. The insert-

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specific part was identical in all primers used for the amplification of the selectable insert (KanaR)

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(forward 5’-AAGGGATTTTGGTCATGAGATTATC, reverse 5’-TGTGCGCGGAACCCCTATTTG). Primers for

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amplification of VP1 had a different insert-specific part (5’-CATCATCATCATCATCACAGCAGC, reverse

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5’-TTAGTTTGCTGGTTTTGCAGGGG). These insert-specific parts of the primers allowed robust

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amplification of the DNA fragments needed for cloning reactions. The second part of each primer

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was the homology region required for SLiCE-mediated homologous recombination. This part varied

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depending on the experimental setup (Supplementary Table 1): First, to study the effect of repeated

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lysate freeze-thaw cycles on the output of SLiCE-mediated cloning, 35 bp-long vector-specific

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sequences of pKM068, neighboring the BamHI recognition site within the ORE1 spacer, were added

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to the KanaR-specific sequences. The same primers were used in the second experimental setup to

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test the effect of different lysate generating and freezing protocols on SLiCE cloning output. In the

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third experimental setup we investigated the output of SLiCE cloning with respect to the

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transformation efficiency of the E. coli cells used for propagation of the reaction products. Therefore,

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35 bp-long vector-specific sequences of pKM068 closely neighboring the BamHI recognition site

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within the ORE1 spacer (distance 10 bp and 50 bp) were added to the KanaR-specific sequences. In

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the fourth experimental setup we investigated the efficiency of SLiCE cloning after linearization of

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ACCEPTED MANUSCRIPT the vector backbone with either one or two REs. We added 35 bp-long vector-specific sequences of

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pYES2/CT directly neighboring BamHI and HindIII recognition sites within the multiple cloning site of

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the vector to the specific VP1 sequences. In the fifth experimental setup we tested the effect of

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different lengths of the homology regions on the output of SLiCE cloning. Therefore, vector-specific

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sequences of pKM068 directly neighboring the BamHI site within the spacer ORE1 were added to the

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insert-specific primer sequences. The lengths of the vector-specific sequences (= homology regions)

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varied from 15 to 95 bp. In the sixth experimental setup we characterized the output of SLiCE-

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mediated homologous recombination cloning in cases where the vector-specific primer sequences

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did not directly neighbor the BamHI site within the ORE1 spacer in pKM068. Homology regions of 35

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bp were designed to perfectly fit the vector sequences 10 - 800 bp distant from the BamHI site within

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the ORE1 segment. In the seventh experimental setup we investigated whether RE recognition sites

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can be inserted 5’ or 3’ of the insert of interest by including the recognition sites into the homology

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regions. Therefore, primers with 36 bp-long vector-specific sequences from the second experiment

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were modified, resulting in 15 bp perfect fit to the vector followed by 6 bp RE recognition site and

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further 15 bp with perfect fit to the vector. These sequences were added to the KanaR-specific

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sequences.

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193 2.4.4 Determination of cloning results

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Different measures were used to characterize the cloning results. First, the number of E. coli colonies

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on agar plates was determined after overnight incubation (cloning efficiency). Second, colony PCR

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was used to amplify the integrated inserts from 5 - 10 colonies, and amplicons were checked by

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electrophoresis on 1% [w/v] agarose gels at 80 - 120 V for 30 - 60 min (cloning accuracy). Third,

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plasmids were isolated from 1 - 10 individual colonies after overnight culture of cells in 3 ml LB

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medium at 37°C and 230 rpm using NucleoSpin Plasmid EasyPure 250 kit (Macherey-Nagel, Düren,

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Germany). Resulting plasmids were sequenced by LGC Genomics (Berlin, Germany).

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2.5 Non-model SLiCE cloning reactions

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Besides the systematic characterization of SLiCE cloning reactions we continuously documented the

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day-to-day performance of SLiCE clonings in different projects. All projects included the insertion of

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one DNA fragment into a vector backbone. Primers for non-model reactions were designed as

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follows: insert-specific parts of the primers were designed solely based on the insert sequence to get

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a melting temperature of 58°C or 60°C calculated with the 4+2 rule (4°C for each G or C, 2°C for each

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A and T). Homology region parts of the primers (always 35 bp) were designed solely based on the

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desired integration site within the vector, irrespective of the sequence at this site. Inserts of interest

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were amplified using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific). The PCR 6

ACCEPTED MANUSCRIPT reaction components were: 50 μl total volume with 1 U polymerase, 10 μl 5 x Phusion HF buffer, 200

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µM dNTPs, 1 ng of plasmid DNA template, and 20 pmol of each primer. The PCR cycling parameters

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were 98°C 30 s, (98°C 10 s, 58°C 30 s, 72°C for 1 min / 2 kb) × 35 cycles, 72°C 5 min, and 4°C infinite.

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PCR products of all inserts were purified via gel extraction before use in SLiCE cloning. Vectors of

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interest were digested with one or two REs of the multiple cloning site. Reactions were performed in

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10 µl volumes with 1 µl SLiCE lysate, 1 µl SLiCE buffer, 3 µl non-purified linearized vector backbone

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and 5 µl insert. For identification of correctly assembled clones, colony PCRs were conducted using

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vector-specific primers to identify clones containing expected fragment sizes. Full sequence identity

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of insert and homology regions compared to planned sequences was verified for one clone per

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construct via plasmid sequencing (LGC Genomics).

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3 Results and discussion

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Cloning of double-stranded DNA fragments into plasmids can be done in several ways. Here, we

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characterized SLiCE-mediated cloning using lysates prepared from the common E. coli strain DH10B,

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which is available in most labs. As SLiCE employs homologous recombination, DNA fragments of

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interest were amplified by polymerase chain reaction (PCR) with primers that included insert-specific

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and vector-specific sequences. The latter, termed homology regions, give the insert the necessary

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overlap to the ends of the vector backbone. Thereafter, inserts were inserted into linearized and

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dephosphorylated vectors by SLiCE and transformed into chemically competent E. coli cells, as

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described in Materials and methods. The colonies formed after overnight incubation were counted to

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determine cloning efficiency (number of all clones) and were further characterized by colony PCR,

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plasmid preparation and sequencing to determine cloning accuracy (number of clones identical to

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the proposed sequence).

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3.1 Effect of freeze-thaw cycles

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The efficiency of SLiCE-mediated cloning is affected by the competency of the bacterial lysates. Zhang

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et al. previously observed that even repeated freeze-thaw cycles did not impair the competency of

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E. coli PPY lysates13. As we employed lysates from a different E. coli strain, DH10B, in our experiments

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we tested whether storage at -80°C or -20°C followed by several freeze-thaw cycles affects cloning

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efficiency. As a marker for cloning success we employed the KanaR insert (kanamycin resistance gene

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including bacterial promoter and terminator) flanked by 35 bp-long homology regions, and BamHI-

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linearized pKM068 vector. Chemically competent E. coli cells were transformed with reaction mixture

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and plated on LB agar containing kanamycin for selection. After overnight incubation the number of

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colonies was determined. We found that repeatedly freezing/thawing bacterial extracts drastically

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reduces cloning efficiency already after the first freeze/thaw cycle; the effect was less pronounced 7

ACCEPTED MANUSCRIPT upon storage at -80°C than at -20°C (Figure 1). A possible reason for the difference between our

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results for E. coli DH10B and the results obtained for E. coli PPY13 might be that SLiCE cloning with

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PPY lysates largely depends on the activity of recombinantly expressed lambda prophage

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recombination proteins while SLiCE cloning with DH10B lysates relies on the activity of endogenous

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bacterial recombination enzymes. Recombinant expression of lambda prophage proteins might result

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in higher concentration of recombination enzymes in the lysates; alternatively, prophage proteins

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might be more tolerant towards repeated freeze-thaw cycles.

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Figure 1. Effect of freeze-thaw cycles on SLiCE cloning efficiency. Bacterial lysates were subjected to

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different numbers of freeze-thaw cycles with lysates stored at -80°C (dark-grey bars) or -20°C (light-

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grey bars). Mean ± SD of three independent experiments are shown. SLiCE reaction: 50 ng modified

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vector backbone, 80 ng selectable insert with 35-bp homology regions, 2 µl of SLiCE reaction

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transformed into 25 µl competent E. coli cells (1 x 109 cfu/µg), 450 µl plated on LB agar with

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kanamycin.

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3.2 Effect of lysate generation and freezing

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The results reported above clearly show a decline of the quality of SLiCE extracts upon repeated

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freezing and thawing. We therefore modified our protocol for shock-freezing freshly prepared SLiCE

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lysates in small (10-µl) aliquots. We compared two different freezing methods: (i) The lysate was

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directly pipetted into liquid nitrogen which resulted in the formation of small frozen lysate beads

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that could be stored thereafter at -80°C. (ii) The bacterial lysate was first aliquoted into 0.2-ml thin-

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walled plastic tubes and then frozen in liquid nitrogen, followed by storage at -80°C. Our data show

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that method (i) supports efficient cloning better than method (ii) (Table 1).

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ACCEPTED MANUSCRIPT In addition, the procedure employed for lysate preparation may affect lysate quality. However, as the

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mechanism of in vitro homologous recombination in E. coli lysates is currently not known in detail,

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defining optimal lysis parameters is challenging. For the preparation of SLiCE lysates, E. coli cells are

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treated with CelLytic B Cell Lysis Reagent. To identify optimal conditions for lysate preparation we

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varied incubation time (10 min to 1 h) and temperature (room temperature, 30°C, and 37°C), and

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tested different ratios of cell pellet amount to lysis reagent volume. However, changing these

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parameters did not improve lysate competency (data not shown).

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We next tested whether freezing the bacterial cell pellet before lysis (overnight storage at -80°C)

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yields lysates with an increased competency for homologous recombination. Cloning reactions

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involved the selectable KanaR insert with 35-bp homology regions and BamHI-linearized pKM068

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vector. Chemically competent E. coli cells were transformed with reaction mixture and plated on LB

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agar containing kanamycin for selection. After overnight incubation the number of colonies was

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determined. Our results show that freezing the bacterial pellet before cell lysis results in a higher

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number of colonies (Table 1).

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Table 1. Effect of lysate generation on SLiCE cloning Fresh pellet for lysis

Exp. 2

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Exp. 3

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Average

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p-value

Lysate into N2

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Number of E. coli colonies obtained for lysates derived from fresh or pre-frozen bacterial pellet,

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frozen either directly in liquid nitrogen or after aliquoting into thin-walled tubes. SLiCE reaction: 50

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ng modified vector backbone, 80 ng selectable insert with 35-bp homology regions, 2 µl of SLiCE

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reaction transformed into 25 µl E. coli (1 x 109 cfu/µg), 450 µl plated on LB agar with kanamycin.

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3.3 Effect of the competency of transformed cells

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Cloning efficiency not only depends on the ligation/recombination method used for joining DNA

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fragments but also on the transformation competency of the E. coli cells. Previously, only highly

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competent cells (2 x 108 - 1 x 109 cfu/µg) were used for SLiCE-mediated cloning14-16. However,

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commercially available highly competent cells are relatively costly, while cells prepared in-house

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often do not reach such high competence levels. We therefore tested whether high competency is

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required for success in SLiCE-mediated cloning. To this end, plasmid pKM068 was linearized with

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ACCEPTED MANUSCRIPT BamHI, dephosphorylated, purified, and used in SLiCE reactions with KanaR as the selectable insert.

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Commercially available and home-made competent cells with different transformation efficiencies

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were transformed with SLiCE reaction mixes and plated on LB agar plates containing kanamycin.

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Colonies were counted after overnight incubation (Table 2). As expected, cells with higher

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competency can result in higher cloning efficiency, although there was no linear correlation between

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the competency of the transformed cells and the number of colonies grown after overnight

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incubation on selection medium. Our results show that self-made chemically competent cells give a

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sufficient number of colonies for cloning of one insert into a vector backbone. In both experimental

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setups (distance of the homology regions to the vector linearization site 10 bp or 50 bp, respectively,

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on both sides), sequencing of ten individual clones each showed a perfect match to the proposed

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sequence. Thus, for SLiCE-mediated cloning of a single DNA fragment into a vector a high level of

309

competency of bacterial cells is not needed; self-made cells are well suited for such cloning

310

experiments.

311

Table 2. Effect of competency of transformed cells on SLiCE cloning DH5-alpha

NEB 5-alpha

50 bp

DH5-alpha

NEB 5-alpha

Exp. 1

77

130

Exp. 1

177

460

Exp. 2

113

480

Exp. 2

95

252

Exp. 3

62

101

Exp. 3

165

421

Average

84

Average

146

378

p-value

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10 bp

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237

0,281

p-value

0,028

Numbers of colonies of three independent experiments using two different sets of primers

314

(annealing at a distance of 10 or 50 bp, respectively, from the vector linearization site) are given.

315

SLiCE reaction: 50 ng modified vector backbone, 80 ng selectable insert, primers with homology

316

regions 10 bp or 50 bp upstream/downstream of the BamHI restriction site, 2 µl of SLiCE reaction

317

transformed into 25 µl competent E. coli cells (DH5-alpha 4 x 107 cfu/µg, or NEB 5-alpha 1 x 109

318

cfu/μg), 150 µl plated on LB agar with kanamycin.

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3.4 Effect of vector linearization

321

We next tested to which extent the procedure used for DNA fragment preparation affects SLiCE-

322

mediated cloning. For homologous recombination, a linearized vector backbone and insert with

323

compatible overhangs are needed. Although linear vector can be obtained by RE digestion or PCR

324

amplification, we focused our analysis on vectors linearized by RE digestion as PCR amplification

325

requires additional primers and bears the risk of inserting base errors. First, we wanted to know

326

whether the number of REs used for vector linearization affects cloning efficiency and accuracy.

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328

simultaneously, dephosphorylated to minimize re-ligation of vector backbone, and purified prior to

329

performing the SLiCE reaction with PCR-amplified and purified VP1 coding sequence17 (capsid viral

330

protein 1 of hamster polyomavirus). PCR on ten colonies from each experimental setup showed that

331

90 - 100% of the clones had the VP1 insert integrated into the vector, irrespective of the RE used for

332

linearization of the vector backbone (Figure 2). Sequencing of plasmids (four from each experimental

333

setup) revealed a perfect match to the proposed sequences. Similar to our in vitro recombination

334

results experiments for in vivo homologous recombination showed highest efficiency in cases where

335

the vector backbone was digested and dephosphorylated18-20. Therefore, only one RE was used for

336

vector linearization followed by dephosphorylation and purification in all further characterization

337

experiments.

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Figure 2. Effect of vector backbone linearization on SLiCE cloning. Agarose gel electrophoresis of

341

colony PCR products with an expected amplicon size of 1,318 bp. Lanes 1-10, linearization of vector

342

backbone with BamHI only prior to the SLiCE reaction; lanes, 11-20 linearization with both, BamHI

343

and HindIII; lanes 21-30, linearization with HindIII only. Lanes ´L´, Hyperladder 1kb (Bioline,

344

Luckenwalde, Germany). SLiCE reaction: 50 ng unmodified vector backbone linearized with indicated

345

restriction enzyme(s), 80 ng insert VP1 (1,245 bp), 2 µl of SLiCE reaction transformed into 25 µl

346

competent E. coli cells (4 x 107 cfu/µg), 150 µl plated on LB agar with carbenicillin.

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3.5 Effect of the lengths of homology regions

349

Like in other overlap-based cloning methods the length of the homology region may affect efficiency

350

and accuracy of SLiCE-mediated clonings. While shorter homology regions (10 - 34 bp) were already

351

tested with E. coli JM109 lysates by Motohashi15, we here used E. coli DH10B lysates and focused on

352

longer homology regions as those are sometimes necessary for the insertion of fragments or

353

mutations not exactly flanking the RE recognition site. We therefore also tested asymmetric

354

combinations of homology regions with different lengths. Selectable KanaR inserts were generated

355

with different lengths of the homology region. Purified PCR products were cloned into BamHI-

356

linearized, dephosphorylated and purified vector backbone (pKM068). After in vitro homologous

357

recombination, chemically competent E. coli cells were transformed with reaction mixture and plated 11

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359

colonies after overnight incubation (Figure 3A). Our results show that symmetric homology regions

360

(same lengths of homology regions at the 5’ and 3’ ends of the insert) as short as 15 bp can lead to

361

successful integration of inserts into linearized vector (see Figure 3B for experimental results

362

obtained by a different researcher). Longer homology regions of up to 45 bp resulted in increased

363

numbers of colonies. A further increase of the length of the homology region from 45 bp to 85 bp did

364

not affect cloning efficiency, while expanding the homology region to 95 bp reduced cloning

365

efficiency. Furthermore, homologous recombination of an insert into linearized vector backbone is

366

also possible with asymmetric homology regions (homology regions on 5’ and 3’ ends of the insert

367

having different sizes) (Figure 3B). In all cases, several of the sequenced clones (generally 10, or less

368

when cloning efficiency was low) showed a perfect match to the proposed sequence, 50 bp up- and

369

downstream of the insertion site. In clones with erroneous sequences we typically observed single

370

base-pair mismatches within the insert, most likely due to errors introduced by Taq polymerase

371

(Maxima Hot Start Green PCR Master Mix) during PCR amplification of the insert. Similar to our

372

results, Zhang et al. showed that 15 bp of homology are sufficient for successful cloning with lysates

373

of E. coli DH10B and PPY, while no colonies formed in experiments with 10 bp-long homology

374

regions13. In contrast, Motohashi demonstrated that even 10 bp-long homology regions allow

375

successful cloning with lysates from E. coli JM10915. Moreover, consistent with our results, both

376

reports showed that increasing the length of the homology regions to up to 52 bp13 or 34 bp15

377

enhances cloning efficiency. In addition to published reports, our data show that cloning efficiency of

378

reactions with asymmetric homology regions can be as successful as reactions with symmetric

379

homology regions, although standard deviations varied considerably between experiments that

380

employed different symmetric homology regions, similar to experiments reported for lysates from E.

381

coli JM10915.

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Figure 3. Effect of symmetric and asymmetric lengths of homology regions on SLiCE cloning. (A)

384

Results for symmetric lengths of homology regions, mean ± SD of three independent experiments.

385

SLiCE reaction: 50 ng modified vector backbone, 80 ng selectable insert, 2 µl of SLiCE reaction

386

transformed into 25 µl competent E. coli cells (1 x 109 cfu/µg), 150 µl plated on LB agar with

387

kanamycin. (B) Results for asymmetric lengths of homology regions, number of colonies of three

388

independent experiments. SLiCE reaction: 50 ng modified vector, 80 ng selectable insert, 4 µl of SLiCE

389

reaction transformed into 50 µl competent E. coli cells (3.8 x 106 cfu/µg), 500 µl plated on LB agar

390

with kanamycin. Experiments for symmetric homology regions in (A) and asymmetric homology

391

regions in (B) were independently performed by two different researchers.

13

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394

For insertion of a DNA fragment into a vector it may be required to remove sequences flanking the

395

restriction site employed for vector linearization. This is particularly relevant if a DNA fragment must

396

be inserted into a vector position where no unique RE site for linearization is close by. In this respect

397

it is important to know whether deleting flanking sequences of different lengths is possible; if so, RE

398

recognition sites further apart from the planned integration site can be employed for fragment

399

insertion. We therefore explored to which extent different combinations of 3’ and 5’ heterologous

400

sequences (ranging in length from 10 to 800 bp) can be deleted from the vector backbone, using

401

primers with homology regions annealing at different distances from the BamHI site of pKM068. As

402

shown in Figure 4, homology regions as distant as 800 bp up- or downstream of the linearization site

403

can be employed for successful insertion of DNA fragments into the vector. In successful insertion

404

reactions the flanking heterologous vector sequences are completely deleted and therefore not

405

present anymore in the resulting construct. We conclude that insertion of DNA fragments into a

406

vector is straightforward with symmetric (Figure 4A) as well as with asymmetric heterologous

407

sequences (Figure 4B).

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Figure 4. Effect of symmetric and asymmetric heterologous vector sequences on SLiCE cloning. (A)

411

Results for symmetric heterologous overhangs of vector backbone, mean value of three independent

412

experiments. SLiCE reaction: 50 ng modified vector backbone, 80 ng selectable insert, primers with

413

homology regions binding to vector sequences distant from the linearization site, 2 µl of SLiCE

414

reaction transformed into 25 µl competent E. coli cells (1 x 109 cfu/µg), 150 µl plated on LB agar with

415

kanamycin. (B) Results for symmetric and asymmetric overhangs of vector backbone, number of

416

colonies of three independent experiments. Experimental design is identical to (A). Experiments for

417

symmetric heterologous sequences in (A) and asymmetric heterologous sequences (B) were done

418

simultaneously.

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As summarized in Table 3, we identified clones with correct sequences for almost every reaction

421

type; in these cases the sequences were as expected 50 bp up- and downstream of the insertion site.

422

Clones with incorrect sequences either contained single base-pair mismatches within the insert

423

sequence, most likely due to errors introduced during PCR, or did not show the expected deletions of 15

ACCEPTED MANUSCRIPT 424

the flanking heterologous regions, most likely resulting from non-homologous end joining of vector

425

backbone and insert.

426 427

Table 3. Sequencing results for SLiCE cloning reactions with flanking heterologous sequences

RI PT

50 bp

100 bp

200 bp

500 bp

800 bp

10 bp

10/10

12/12

12/12

9/10

4/4

3/3

50 bp

12/12

10/10

12/12

4/6

0

5/6

100 bp

11/12

12/12

10/10

8/10

3/3

1/1

200 bp

6/6

6/6

7/9

10/10

3/6

0

500 bp

4/4

1/1

1/3

0

5/10

1/1

800 bp

1/4

0/1

0/5

0

0/1

8/8

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Inserts were sequenced from E. coli colonies obtained in three independent experiments performed

429

to test the effect of symmetric and asymmetric heterologous vector sequences on SLiCE cloning.

430

Given is the ratio of the number of clones with a correct sequence to the number of all clones

431

sequenced. SLiCE reaction: 50 ng modified vector backbone, 80 ng selectable insert, primers with

432

homology regions binding to vector sequences distant from the linearization site, 2 µl of SLiCE

433

reaction transformed into 25 µl competent E. coli cells (1 x 109 cfu/µg), 150 µl plated on LB agar with

434

kanamycin.

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Deletion of flanking heterologous sequences has been shown for a few examples in experiments with

437

E. coli PPY SLiCE lysates, but a systematic study was not performed13; with respect to E. coli JM109

438

SLiCE lysates15 only deletions of 0 bp or 40 bp of symmetric and asymmetric flanking heterologous

439

sequences were reported. A common feature of our and the previous results is that cloning efficiency

440

and accuracy generally benefit from an increased length of the flanking heterologous sequences. In

441

addition, we observed that cloning efficiency is higher with symmetric than asymmetric flanking

442

sequences. Considering our findings we conclude that removal of 5’ and 3’ heterologous sequences

443

of up to 800 bp is possible with E. coli DH10B lysate, although efficiency drops when flanking

444

sequences exceed 100 bp. Other in vitro overlap-based cloning methods like Gibson assembly (NEB)

445

or NEBuilder HiFi DNA Assembly Master Mix (NEB) have no or limited capacity (less than 10 bp),

446

respectively, to remove flanking sequences. The only competitor in this respect is transformation

447

assisted recombination (TAR) cloning in yeast12, an in vivo method that also allows for DNA cloning

448

and the concomitant removal of heterologous vector sequences. However, TAR is considerably more

449

time consuming than SLiCE-mediated cloning and limited to vectors containing a yeast origin of

450

replication and a yeast auxotrophic marker.

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ACCEPTED MANUSCRIPT 451 3.7 Integration of restriction enzyme recognition sites

453

The usability of SLiCE-mediated cloning would further increase if mutations, e.g. to create RE

454

recognition sites for subsequent cloning events, could be introduced in the homology regions

455

flanking the inserted DNA fragment. We therefore analyzed whether RE recognition sites can be

456

introduced into the flanking overlap segments. We selected seven different 6-mer cutters to test for

457

sequence flexibility and integrated their respective recognition sites into the middle of 35-bp long

458

homology regions. We used selectable KanaR inserts with homology regions containing the RE

459

recognition sites upstream or downstream of the inserted DNA fragment. The number of colonies on

460

agar plates was counted after overnight incubation and ten plasmids from each of the 14 clonings

461

were checked by RE digestion (see Figure 5A, B for examples). In each case, four to nine clones had

462

correctly integrated the RE recognition site (Figure 5C). Three randomly selected clones with correct

463

fragmentation patterns were checked by sequencing and in all 42 cases a perfect match to the

464

proposed sequences was observed.

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Figure 5. Integration of restriction enzyme recognition sites into homology regions. Control digests of

467

plasmids derived from SLiCE reactions with RE recognition sites included in the homology regions. (A)

468

NaeI site included in the upstream homology region. (B) NaeI site included in the downstream

469

homology region. SLiCE reaction: 50 ng modified vector backbone, 80 ng selectable insert, 2 µl of

470

SLiCE reaction transformed into 25 µl competent E. coli cells (1 x 109 cfu/µg), 150 µl plated on LB agar

471

with carbenicillin and kanamycin. Examples of restriction patterns after agarose gel electrophoresis

472

are shown. Plasmids were prepared from ten clones each and digested with NaeI. In addition to the

473

NaeI site in the primer´s homology region, two further NaeI sites are located in the insert and vector

474

backbone, giving three fragments upon full digestion for correctly assembled plasmids. Expected

475

fragment lengths are 300/1,080/6,170 bp for (A) and 785/1,338/5,446 bp for (B). Asterisks indicate 17

ACCEPTED MANUSCRIPT 476

positive clones. In (B), fragments of about 2.1 kb represent partial digests. Lanes ´L´, Hyperladder 1kb

477

(Bioline). (C) Number of colonies obtained and number of clones with correct restriction pattern.

478 3.8 Non-model cloning reactions

480

Next we were interested to learn about the performance, robustness, and applicability of SLiCE-

481

mediated cloning for use in daily experimental work. Here, we summarize our observations made

482

with 50 non-model cloning reactions performed over a period of a year, with different DNA

483

fragments and target vectors of the following types: (i) integration of short tags into protein

484

expression vectors (inserts ~100 bp), or (ii) integration of protein coding sequences into vector

485

backbones with multiple cloning sites between promoter and terminator sequences (inserts >500

486

bp). Sizes of vector backbones and insert fragments, as well as the GC content of the homology

487

regions (35 bp each), are given in Table 4. Colony PCRs were conducted using vector-specific primers

488

to identify clones containing fragments expected for correctly assembled inserts (Table 4). One clone

489

per construct was selected and correct sequences of insert and homology regions were confirmed by

490

Sanger sequencing. Our results revealed successful cloning of a wide variety of constructs with

491

backbone sizes ranging from 2,974 bp to 13,548 bp, insert fragment sizes ranging from 91 bp to

492

1,664 bp, and a GC content of the homology regions between 17% and 60%. Notably, there were no

493

obvious relationships between cloning efficiency and vector size or GC content of the homology

494

regions. However, there was a tendency for reduced cloning efficiency of short DNA fragments

495

(Table 4). Further experiments showed that SLiCE-mediated cloning of inserts of 100 - 500 bp (e.g.

496

promoters and terminators) is straightforward with similar success rates (data not shown).

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497

Table 4. Non-model cloning of 50 different constructs

498

Fragment sizes Homology regions Colony PCR Construct Fragment sizes Homology regions Colony PCR Backbone Insert Upstream Downstream Backbone Insert Upstream Downstream Number bp bp GC in % GC in % Correct size Number bp bp GC in % GC in % Correct size 1 2974 1221 47 36 10/10 26 5935 1248 61 53 8/8 2 5136 506 60 57 4/8 27 6440 853 31 51 7/8 3 5136 778 60 51 2/8 28 6440 853 31 51 3/8 4 5360 645 20 54 8/8 29 7047 91 31 46 2/8 5 5360 672 20 54 8/8 30 7047 91 43 17 1/56 6 5493 645 20 54 8/8 31 7047 97 31 46 1/8 7 5493 672 20 54 8/8 32 7047 97 43 17 2/88 8 5739 663 31 17 8/16 33 7649 702 23 17 1/16 9 5739 764 46 34 3/4 34 7649 740 34 26 4/8 10 5739 772 31 51 4/16 35 8351 740 34 26 8/8 11 5739 795 31 51 2/8 36 9759 775 23 20 2/3 12 5739 814 31 51 6/16 37 9759 859 23 57 7/8 13 5739 856 31 17 4/6 38 10464 750 23 43 6/8 14 5739 1124 46 33 4/8 39 10464 859 23 57 3/8 15 5739 1205 46 33 5/8 40 10548 750 23 43 4/8 16 5739 1226 46 33 5/8 41 10548 775 23 20 6/8 17 5739 1256 46 33 8/8 42 10649 702 23 17 1/8 18 5739 1538 46 33 4/8 43 10649 740 34 26 8/8 19 5739 1664 46 33 3/48 44 11351 740 34 26 8/8 20 5840 663 26 51 8/8 45 12759 775 23 20 2/2 21 5840 772 26 51 3/8 46 12759 859 23 57 8/8 22 5840 795 26 51 5/8 47 13464 750 23 43 8/8 23 5840 814 26 51 2/8 48 13464 859 23 57 7/8 24 5935 705 61 53 8/8 49 13548 750 23 43 8/8 25 5935 1221 61 53 8/8 50 13548 775 23 20 1/8 Results of non-model SLiCE cloning reactions performed with inserts and vectors of different sizes, and homology regions (35 bp each) varying in their GC

499

content.

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ACCEPTED MANUSCRIPT 500 4 Conclusions

502

SLiCE is a simple, low-cost and efficient method for seamless and sequence-independent DNA cloning

503

using in vitro homologous recombination. It does not require particular E. coli strains (standard RecA-

504

deficient strains are sufficient) or expensive laboratory devices and is therefore easily established in

505

labs with basic molecular biology equipment. Although the details of the molecular mechanisms of in

506

vitro homologous recombination in E. coli lysates are currently unknown, our systematic analysis

507

performed here with E. coli DH10B lysates revealed a high robustness of the SLiCE method against

508

various experimental parameters (Table 5) including: a virtual absence of length restrictions for the

509

vector backbone, differences of GC content in homology regions, deletion of unwanted vector

510

sequences concomitant with fragment insertion, and changing sequences within homology regions.

511

We further found that freezing the E. coli DH10B cell lysate in small aliquots directly in liquid nitrogen

512

improves cloning efficiency and simplifies experimental handling compared to other freezing

513

protocols.

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Table 5. Summary of aspects investigated and major findings

Major findings

Effect of lysate freeze-thaw cycles

TE

Effect of lysate generation and freezing

D

Aspects investigated

Avoid freeze-thaw cycles of lysate Produce lysate from frozen cell pellets; freeze lysate rapidly; store lysate at -80°C In-house-made cells are sufficient

Effect of vector linearization

One RE is sufficient

Effect of length of homology region

15 bp is sufficient, 25 bp to 35 bp are optimal

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Effect of competency of transformed cells

Effect of heterologous vector sequences

10 bp to 800 bp can be deleted; symmetric deletions are preferred

Integration of RE recognition sites

Possible

Non-model cloning reactions

SLiCE is a robust all-day cloning method

516 517

Despite its simplicity and robustness, SLiCE-mediated cloning has so far not been widely adopted by

518

research laboratories; since the original publication of SLiCE in 2012 only six labs reportedly used

519

SLiCE for cloning21-26. Two other labs used different E. coli strains15 or optimized protocols for lysate

520

production16, and employed vectors equipped with CcdB cassettes for improvement of cloning

521

accuracy27. We envisage that the systematic characterization of SLiCE cloning parameters reported

522

here will attract more researchers in the future to employ SLiCE for their own standard molecular or

20

ACCEPTED MANUSCRIPT 523

synthetic biology projects.

524 Author contributions

526

B. M.-R. initiated the Cell2Fab project and the overall research strategy. K.M. designed the details of

527

the current study and supervised the group. L.H., D.D. and K.S. acquired the data. K.M. analyzed and

528

interpreted the data. K.M. and B.M.-R. wrote the manuscript.

529 530

Notes

531

The authors declare no competing financial interest.

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Acknowledgements

534

We thank Katja Hanack (University of Potsdam, Germany) for plasmid pKP3, and Stefan Rünger and

535

Miriam Rathsmann for technical support provided during practicals. B.M.-R. thanks the Federal

536

Ministry of Education and Research of Germany for funding (Cell2Fab; FKZ 031A172).

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537 Abbreviations

539

RE, restriction enzyme; SLiCE, seamless ligation cloning extract; KanaR, kanamycin resistance gene

540

including bacterial promoter and terminator; TAR, transformation-assisted recombination

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538

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541 References

543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565

[1] Kitney, R., and Freemont, P. (2012) Synthetic biology - the state of play, FEBS Lett. 586, 20292036. [2] Nguyen, Q. T., Merlo, M. E., Medema, M. H., Jankevics, A., Breitling, R., and Takano, E. (2012) Metabolomics methods for the synthetic biology of secondary metabolism, FEBS Lett. 586, 2177-2183. [3] Lee, J. W., Kim, H. U., Choi, S., Yi, J., and Lee, S. Y. (2011) Microbial production of building block chemicals and polymers, Curr. Opin. Biotechnol. 22, 758-767. [4] Westfall, P. J., Pitera, D. J., Lenihan, J. R., Eng, D., Woolard, F. X., Regentin, R., Horning, T., Tsuruta, H., Melis, D. J., Owens, A., Fickes, S., Diola, D., Benjamin, K. R., Keasling, J. D., Leavell, M. D., McPhee, D. J., Renninger, N. S., Newman, J. D., and Paddon, C. J. (2012) Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin, Proc. Natl. Acad. Sci. U.S.A. 109, E111-118. [5] Cohen, S. N., Chang, A. C., Boyer, H. W., and Helling, R. B. (1973) Construction of biologically functional bacterial plasmids in vitro, Proc. Natl. Acad. Sci. U.S.A. 70, 3240-3244. [6] Lu, Q. (2005) Seamless cloning and gene fusion, Trends Biotechnol. 23, 199-207. [7] Ellis, T., Adie, T., and Baldwin, G. S. (2011) DNA assembly for synthetic biology: from parts to pathways and beyond, Integr Biol (Camb) 3, 109-118. [8] Shetty, R. P., Endy, D., and Knight, T. F., Jr. (2008) Engineering BioBrick vectors from BioBrick parts, J Biol Eng 2, 5. [9] Anderson, J. C., Dueber, J. E., Leguia, M., Wu, G. C., Goler, J. A., Arkin, A. P., and Keasling, J. D. (2010) BglBricks: A flexible standard for biological part assembly, J Biol Eng 4, 1-1. [10] Engler, C., Kandzia, R., and Marillonnet, S. (2008) A One Pot, One Step, Precision Cloning Method with High Throughput Capability, PloS One 3.

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SC

RI PT

[11] Gibson, D. G. (2011) Enzymatic Assembly of Overlapping DNA Fragments, In Synthetic Biology, Pt B: Computer Aided Design and DNA Assembly, pp 349-361. [12] Kouprina, N., and Larionov, V. (2006) Innovation - TAR cloning: insights into gene function, longrange haplotypes and genome structure and evolution, Nat. Rev. Genet. 7, 805-812. [13] Zhang, Y., Werling, U., and Edelmann, W. (2012) SLiCE: a novel bacterial cell extract-based DNA cloning method, Nucleic Acids Res. 40, e55. [14] Zhang, Y., Werling, U., and Edelmann, W. (2014) Seamless Ligation Cloning Extract (SLiCE) cloning method, Methods Mol. Biol. 1116, 235-244. [15] Motohashi, K. (2015) A simple and efficient seamless DNA cloning method using SLiCE from Escherichia coli laboratory strains and its application to SLiP site-directed mutagenesis, BMC Biotechnol. 15. [16] Okegawa, Y., and Motohashi, K. (2015) Evaluation of seamless ligation cloning extract preparation methods from an Escherichia coli laboratory strain, Anal. Biochem. 486, 51-53. [17] Messerschmidt, K., Hempel, S., Holzlohner, P., Ulrich, R. G., Wagner, D., and Heilmann, K. (2012) IgA antibody production by intrarectal immunization of mice using recombinant major capsid protein of hamster polyomavirus, Eur J Microbiol Immunol (Bp) 2, 231-238. [18] Bubeck, P., Winkler, M., and Bautsch, W. (1993) Rapid Cloning by Homologous Recombination in-Vivo, Nucleic Acids Res. 21, 3601-3602. [19] Oliner, J. D., Kinzler, K. W., and Vogelstein, B. (1993) In-Vivo Cloning of Pcr Products in Escherichia-Coli, Nucleic Acids Res. 21, 5192-5197. [20] Parrish, J. R., Limjindaporn, T., Hines, J. A., Liu, J. Y., Liu, G. Z., and Finley, R. L. (2004) Highthroughput cloning of Campylobacter jejuni ORFs by in vivo recombination in Escherichia coli, J Proteome Res. 3, 582-586. [21] Hornsby, M., Paduch, M., Miersch, S., Saeaef, A., Matsuguchi, T., Lee, B., Wypisniak, K., Doak, A., King, D., Usatyuk, S., Perry, K., Lu, V., Thomas, W., Luke, J., Goodman, J., Hoey, R. J., Lai, D., Griffin, C., Li, Z., Vizeacoumar, F. J., Dong, D., Campbell, E., Anderson, S., Zhong, N., Graeslund, S., Koide, S., Moffat, J., Sidhu, S., Kossiakoff, A., and Wells, J. (2015) A High Through-put Platform for Recombinant Antibodies to Folded Proteins, Mol. Cell Proteomics 14, 2833-2847. [22] Peykov, S., Berkel, S., Schoen, M., Weiss, K., Degenhardt, F., Strohmaier, J., Weiss, B., Proepper, C., Schratt, G., Noethen, M. M., Boeckers, T. M., Rietschel, M., and Rappold, G. A. (2015) Identification and functional characterization of rare SHANK2 variants in schizophrenia, Mol. Psychiatry 20, 1489-1498. [23] Brown, W. R. A., Thomas, G., Lee, N. C. O., Blythe, M., Liti, G., Warringer, J., and Loose, M. W. (2014) Kinetochore assembly and heterochromatin formation occur autonomously in Schizosaccharomyces pombe, Proc. Natl. Acad. Sci. U.S.A. 111, 1903-1908. [24] Litzlbauer, J., Schifferer, M., Ng, D., Fabritius, A., Thestrup, T., and Griesbeck, O. (2015) Large Scale Bacterial Colony Screening of Diversified FRET Biosensors, PloS One 10. [25] Inobe, T., and Genmei, R. (2015) N-Terminal Coiled-Coil Structure of ATPase Subunits of 26S Proteasome Is Crucial for Proteasome Function, PloS One 10. [26] Inoue, H., Suzuki, D., and Asai, K. (2013) A putative bactoprenol glycosyltransferase, CsbB, in Bacillus subtilis activates SigM in the absence of co-transcribed YfhO, Biochem. Biophys. Res. Commun. 436, 6-11. [27] Zhang, P., Du, E., Ma, J., Wang, W., Zhang, L., Tikoo, S. K., and Yang, Z. (2015) A Novel and Simple Method for Rapid Generation of Recombinant Porcine Adenoviral Vectors for Transgene Expression, PloS One 10.

AC C

566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612

22

ACCEPTED MANUSCRIPT

Supplementary Table 1. Sequences of primers used in this work Sequence 5' => 3'

Orientation

IP T

Usage

Effect of freeze-thaw cycles GAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

CR

GCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG Effect of lysate generation and freezing GAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC Effect of competence of transformed cells 10 bp

GACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTC AAGGGATTTTGGTCATGAGATTATC

10 bp

GTCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTGTGCGCGGAACCCCTATTTG

50 bp

CACGTGACCTGCTTCTCCGACCAAGAAACCGAAGAC AAGGGATTTTGGTCATGAGATTATC

50 bp

GACCGTCGAAAGGTTTGCCGGAGAATTGAGTTTCTTTGTGCGCGGAACCCCTATTTG

Effect of vector linearization

M AN US

GCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

D

TAGCAGCTGTAATACGACTCACTATAGGGAATATTATG CATCATCATCATCATCACAGCAGC TGCAGAATTCCAGCACACTGGCGGCCGTTACTAGTTTAGTTTGCTGGTTTTGCAGGGG

TE

Effect of length of homology region

forward reverse forward reverse forward reverse forward reverse forward reverse

TTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

15 bp

TCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

25 bp

ACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

25 bp

CGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

35 bp

GAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

35 bp

GCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

45 bp

ACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

45 bp

TCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

55 bp

AAGACAAAAGACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

55 bp

TTCTTGACCGTCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

65 bp

CAAGAAACCGAAGACAAAAGACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

65 bp

AGAATTGAGTTTCTTGACCGTCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

EP

15 bp

AC C

613

23

forward reverse

ACCEPTED MANUSCRIPT

CTTCTCCGACCAAGAAACCGAAGACAAAAGACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

75 bp

GGTTTGCCGGAGAATTGAGTTTCTTGACCGTCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

85 bp

ACGTGACCTGCTTCTCCGACCAAGAAACCGAAGACAAAAGACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

85 bp

ACCGTCGAAAGGTTTGCCGGAGAATTGAGTTTCTTGACCGTCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

95 bp

TCGTCGTCTCACGTGACCTGCTTCTCCGACCAAGAAACCGAAGACAAAAGACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC forward

95 bp

ACGAATCACGACCGTCGAAAGGTTTGCCGGAGAATTGAGTTTCTTGACCGTCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

CR

IP T

75 bp

Effect of heterologous vector sequences

reverse

GACTTGTCCACGAGTCCAAAGACGGTTTTGGTTCTCAAGGGATTTTGGTCATGAGATTATC

10 bp

GTCAAGCAACAGCTTCATTAGCGAATAATTGTCTTGTGTGCGCGGAACCCCTATTTG

50 bp

CACGTGACCTGCTTCTCCGACCAAGAAACCGAAGACAAGGGATTTTGGTCATGAGATTATC

50 bp

GACCGTCGAAAGGTTTGCCGGAGAATTGAGTTTCTTTGTGCGCGGAACCCCTATTTG

100 bp

CTTCGTTAATGGATTGTTCTCAACGAGACTCCTTCAAAGGGATTTTGGTCATGAGATTATC

100 bp

AACTCAGAAATTCCAAACGCAATCCAATTCTTCTGTTGTGCGCGGAACCCCTATTTG

200 bp

TGGGTTATATGTCGTGTTTTCCAAAAACGTGCCGATAAGGGATTTTGGTCATGAGATTATC

200 bp

TAGCATATTTTTTTCTCCTTTTTTACAAAAAAATGTTGTGCGCGGAACCCCTATTTG

500 bp

AAACCGGGCGACAGAAGCCGGTTATTGGAAAGCCACAAGGGATTTTGGTCATGAGATTATC

forward

500 bp

GTACACGCGTCTGTACAGAAAAAAAAGAAAAATTTGTGTGCGCGGAACCCCTATTTG

reverse

800 bp

CTGATGAAGAACTCATAACTCACTACCTCAAACCAAAAGGGATTTTGGTCATGAGATTATC

forward

800 bp

CGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTGTGCGCGGAACCCCTATTT

reverse

Integration of restriction enzyme recocgnition sites with SLiCE

EP

TE

D

M AN US

10 bp

forward reverse forward reverse forward reverse forward reverse

GAGTCCAAAGACGGTTTTGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

w/o

GCTTCATTAGCGAATAATTGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

EcoRI

GAGTCCAAAGACGGGAATTCTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

EcoRI

GCTTCATTAGCGAAGAATTCTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

NcoI

GAGTCCAAAGACGGCCATGGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

NcoI

GCTTCATTAGCGAACCATGGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

NdeI

GAGTCCAAAGACGGCATATGTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

NdeI

GCTTCATTAGCGAACATATGTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

NheI

GAGTCCAAAGACGGGCTAGCTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

NheI

GCTTCATTAGCGAAGCTAGCTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

AC C

w/o

24

ACCEPTED MANUSCRIPT

GAGTCCAAAGACGGGCCGGCTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

NaeI

GCTTCATTAGCGAAGCCGGCTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

reverse

BglII

GAGTCCAAAGACGGAGATCTTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

forward

BglII

GCTTCATTAGCGAAAGATCTTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

SacI

GAGTCCAAAGACGGGAGCTCTTCTCTGTTTTACTCAAGGGATTTTGGTCATGAGATTATC

SacI

GCTTCATTAGCGAAGAGCTCTCTTGTAAAAACAGATGTGCGCGGAACCCCTATTTG

CR

IP T

NaeI

AC C

EP

TE

D

M AN US

614

25

reverse forward reverse