protocol

Engineering complex biological systems in bacteria through recombinase-assisted genome engineering Christine Nicole S Santos1,4 & Yasuo Yoshikuni1–3 1Bio Architecture Lab, Inc., Berkeley, California, USA. 2BALChile S.A., Santiago, Chile. 3BAL Biofuels S.A., Santiago, Chile. 4Present address: Manus Biosynthesis, Inc.,

Cambridge, Massachusetts, USA. Correspondence should be addressed to Y.Y. ([email protected]).

© 2014 Nature America, Inc. All rights reserved.

Published online 15 May 2014; doi:10.1038/nprot.2014.084

Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. This engineering strategy was previously applied to the development of an Escherichia coli–based platform, which enabled the use of brown macroalgae as a feedstock for the production of biofuels and renewable chemicals. In this approach, functional genetic modules are first designed in silico and constructed on a bacterial artificial chromosome (BAC) by using a recombineering-based inchworm extension technique. Stable integration into the recipient chromosome is then mediated through the use of recombinase-assisted genome engineering (RAGE). The flexibility, simplicity and speed of this method enable a comprehensive optimization of several different parameters, including module configuration, strain background, integration locus, gene copy number and intermodule compatibility. This paradigm therefore has the potential to markedly expedite most strain-engineering endeavors. Once a biological system has been designed and constructed on a BAC, its implementation and optimization in a recipient host can be carried out in as little as 1 week.

INTRODUCTION Over the past few decades, we have seen a substantial surge in the use of prokaryotic systems, such as E. coli, for a myriad of applications, from elucidating gene and cellular function (functional genomics, genetic circuits)1–4 to the development of microbial cell factories for the production of renewable fuels and chemicals5–8. Indeed, the availability of a wide array of tools for novel pathway discovery and elucidation9, de novo DNA synthesis10,11, genetic parts assembly12–15 and studies on transcriptional and translational control16–19 has enabled the engineering of larger and increasingly complex biological systems. With this complexity, however, comes uncertainty and unpredictability, and as a result, most engineered systems still require several rounds of troubleshooting in order to attain optimal performance in a microbial host. Because of the oftentimes laborious nature of this optimization process and the limited capabilities of existing genome engineering tools, many studies to date still rely on the use of plasmids for gene and pathway expression. However, it is now generally well understood that these systems suffer from genetic (and hence phenotypic) instability related to plasmid loss, allele inactivation, copy number variability and plasmid-associated metabolic burden20–24. As such, there has been renewed interest in the design, construction and implementation of stable, chromosomally integrated biological systems to ensure robust and reproducible phenotypic performance. Recently we reported the development of an efficient and flexible RAGE approach for the stable implementation and optimization of large, complex biological systems in microbial organisms25,26. Application of this strain-engineering paradigm led to the construction of an E. coli–based platform for the production of biofuels and renewable chemicals using brown macro­ algae as feedstock. Brown macroalgae possess several desirable properties, which make them ideal next-generation feedstock for biofuel and chemical production. Unlike land-based feedstock such as sugarcane or corn, seaweed cultivation does not require arable land, fertilizer or fresh water resources, thus minimizing

1320 | VOL.9 NO.6 | 2014 | nature protocols

its impact on both the environment and existing food supplies. Pretreatment requirements for seaweed are also minimal due to the absence of lignin. However, realizing the economic potential of seaweed has so far been hampered by the inability of industrial microbes to digest and metabolize alginate, one of the main sugar components in seaweed. Engineering this unique property into an E. coli platform required simultaneous implementation and parallel optimization of three separate modules: (i) an alginate lyase secretion machinery, (ii) the alginate uptake and catabolism pathway and (iii) the homoethanol pathway. As outlined in Figure 1, this strain-engineering paradigm can be roughly divided into four main stages: design, construction, implementation and troubleshooting. In the design stage, the general blueprint for a biological system is created by determining the appropriate functional modules (either naturally occurring or artificial assemblies) required for a particular property of interest. These modules are then constructed and incorporated into a single-copy plasmid or BAC through direct cloning, overlap PCR followed by recombineering (which is the method described in this protocol; see the enlarged middle panel in ‘construction’ in Fig. 1), or multigene or multipart assembly10–15,27. In the next stage, implementation of the biological system is accomplished through validation of the BAC-residing system in the desired host(s) followed by the use of site-specific recombinases (such as the Cre or FLP recombinases) to mediate module insertion into predetermined sites within the chromo­some. Notably, the relative simplicity and speed of this step enables one to undertake a multipronged approach to study the effects of several parameters (e.g., genetic background, integration loci, gene copy number and intermodule interactions or compatibility) on cellular phenotype. If additional optimization is required, troubleshooting can be performed through iterative rounds of design, construction, and implementation, or, alternatively, through complementary genome editing or adaptive evolutionary strategies8,22,28.

Use of single-copy plasmids or BACs to mimic genome-based expression. The use of multicopy plasmids for pathway expression and subsequent strain optimization is still very common. Unfortunately, however, optimized phenotypes are rarely retained once constructs are transferred into the chromosome, thus requiring additional rounds of troubleshooting and testing. To bridge this inherent gap between multicopy plasmid-based and genome-based phenotypes, our methodology makes use of single-copy plasmids or BACs for initial pathway construction and characterization. In doing so, we are able to exploit genetic tools for the manipulation and assembly of plasmids, all while using a system that is representative of genome-based expression. In addition, BACs can easily accommodate a large amount of genetic material (typically up to 300 kb long), as would be required for complex, multimodule biological systems29. Practically speaking, this means that the performance of a given design can be roughly validated and, to a certain degree, optimized on a BAC before being incorporated into the recipient bacterial chromosome. Use of site-specific recombinases for genome engineering. Although site-specific recombinases are extensively used for chromosomal integration in eukaryotic systems, their use in prokaryotes such as E. coli has so far been limited to simple gene deletion applications such as marker excision or recycling30–33. However, as we demonstrated in a recent report, pairing these recombinases with sets of mutually exclusive (i.e., noninteracting yet selfrecognizing) target sites presents a powerful tool for prokaryotic genome engineering, particularly for the incorporation of large and complex biological systems25. Indeed, no other single genome-editing tool offers the efficiency, accuracy, specificity and flexibility afforded by RAGE. For example, although homologous recombination of single- or double-stranded DNA fragments (‘recombineering’) has been frequently used for chromosomal integration, efficiencies of recombination drop substantially for cassette sizes over just a few kilobases long, thus precluding its use for larger systems32,34,35. In addition, the requirement for PCR amplification to generate linear DNA templates imposes further size limitations and provides an undesirable entry point for the introduction and propagation of sequence errors. In contrast, RAGE does not show a considerable drop in integration efficiencies for larger cassette sizes and, in our hands, it has been experimentally validated for the single-step incorporation of a 59-kb-long multimodule pathway. The true upper bound for fragment insertion is probably closer to 300 kb, a limit imposed by the capacity of the BAC. Recombination with RAGE is also

Construction (Steps 4–35)

Microbe A Microbe B Microbe C Microbe D

Promoters RBS etc.

Overlap PCR DNA fragments Recombineering Direct cloning

Gene assembly

Transformation into different heterologous hosts

Implementation through RAGE (Steps 36–54)

© 2014 Nature America, Inc. All rights reserved.

Advantages of our method This strain-engineering paradigm incorporates two main elements, which greatly simplify and expedite the stable implementation and optimization of particularly complex biological systems.

Phenotypic screening Chromosomal integration

Varying integration loci

Varying copy number

Parallel optimization of multiple modules

Phenotypic screening Troubleshooting

Figure 1 | Strain-engineering design principle based on the use of RAGE for the optimal implementation of complex biological systems. Specific activities fall into four main stages: design, construction, implementation and troubleshooting. MAGE, multiplex automated genome engineering.

Design (Steps 1–3)

protocol

Directed or adaptive evolution, such as MAGE

highly accurate, as integration is mediated directly between the BAC template and the genome without PCR amplification, thus minimizing sequence errors. As an alternative to recombineering, phage integrase–based methods (recombination between attP and attB sites) offer higher integration efficiencies and the ability to handle large donor fragments36,37. However, the presence of existing att sites within the recipient bacterial chromosome affords little flexibility with respect to the specific site(s) of integration. In addition, phage integration often leads to the incorporation of the entire donor plasmid, including potentially undesirable components on the vector backbone, such as the origin of replication and antibiotic selection markers. Here again, RAGE is distinguished by its flexibility and specificity through its ability to direct the desired genetic elements into any location within the genome (through strategic placement of the recombinase recognition sites). Notably, this method is flexible not only with respect to integration loci but also with respect to the donor template; although we focus on the use of BACs in this protocol, RAGE implementation can, in fact, accommodate other donor sources, including multicopy plasmids and genomic DNA. nature protocols | VOL.9 NO.6 | 2014 | 1321

protocol Table 1 | Functional modules in the microbial platform for ethanol production from brown macroalgae. Evaluation

© 2014 Nature America, Inc. All rights reserved.

Modules

Inputs

Outputs

Secretable alginate lyase

Na-alginate (Sigma-Aldrich, cat. no. A2158)

Oligoalginate formation measured by OD254

Oligoalginate metabolism   pathway

Oligoalginate made from Na-alginate treated with alginate lyase (Sigma-Aldrich, cat. no. A1603)

Cell growth measured by OD600

Ethanol production pathway

d-Dextrose

Ethanol and byproduct formation measured by HPLC

(Sigma-Aldrich, cat. no. G8270) d-Mannitol (Sigma-Aldrich, cat. no. D4125) d-Galacturonic acid sodium salt (Sigma-Aldrich, cat. no. 73960)

Altogether, merging these two elements allows one to substantially expedite strain-engineering efforts. The use of single-copy plasmids or BACs minimizes unexpected complications arising from plasmid-to-genome transfers, and integration via RAGE greatly facilitates further optimization at the genome level. The simplicity and speed of this approach also allow for substantial parallelization, thus making it possible to explore the effects of several module configurations (e.g., orthologous enzymes and/or pathways and different expression levels as controlled by transcriptional and translational elements) and integration parameters (e.g., strain background, integration loci, gene copy number and intermodule interactions or compatibility) simultaneously. We note that although this protocol focuses on the application of this method specifically to E. coli, the concepts and techniques described here can be easily translated to other prokaryotic systems and perhaps to higher organisms such as yeast as well. Experimental design In this protocol, we provide one example of how our strain­engineering paradigm can be applied to the design, construction, implementation and troubleshooting of a complex biological system in E. coli. Design (Steps 1–3). In designing a biological system, it is often helpful to first divide it into a few modules comprising smaller and less-complex systems. Ideally, each of these modules is designed to be functionally independent of the others in order to enable easy characterization and optimization of module performance through an available input and an easily quantified output. In addition, it is also crucial to define functional boundaries in a way that minimizes interaction or interference between modules apart from their inputs and outputs. For example, in developing an E. coli platform for the conversion of brown macroalgae to biofuels and other renewable chemical compounds, we first divided the system into three distinct functional modules: (i) a secretable alginate lyase (for degradation of the unique sugar polymer, alginate), (ii) the oligoalginate uptake and metabolism pathway (transporters and metabolic pathways that convert oligoalginate into pyruvate and glyceraldehyde-3-phosphate) and (iii) the pathway for ethanol production. These boundaries were defined on the basis of the aforementioned criteria as described in Table 1. 1322 | VOL.9 NO.6 | 2014 | nature protocols

Once module boundaries are defined, each module is then designed independently. For metabolic pathways, which already exist in other bacterial hosts, it is often more straightforward to directly recruit naturally occurring pathways and enzymes and transfer them into more tractable hosts such as E. coli. This is particularly the case for pathways related to growth and survival, as evolutionary pressures have, to a certain degree, already preoptimized enzymatic properties (kinetics, substrate specificity, reaction selectivity and stability) and pathway expression to grant their natural hosts an improved fitness advantage. Genes encoding related pathway components are often found in close physical proximity to each other in the bacterial genome, with their expression coordinated by the same regulatory element(s). As such, many important characteristics of optimal pathways can be transferred into the desired host through incorporation of whole genomic segments into these modules. If no natural microbial pathway or source exists for a specific function or if additional manipulation is needed to obtain optimal performance in the desired host strain, modules can be designed and assembled by traditional synthetic biology approaches. Here sophisticated design elements for controlling expression at the transcriptional and translational levels can be incorporated to allow for finetuning of both native and heterologous pathways16–19. For engineering alginate use by E. coli, we initially sought all necessary genetic components for alginate degradation and metabolism from the National Center for Biotechnology Information (NCBI) database. Because the molecular mechanism for alginate degradation and catabolism had not been fully elucidated, we deciphered it in the marine microbe Vibrio splendidus 12B01 on the basis of the mechanism for pectin degradation and metabolism in the plant pathogenic enterobacterium, Erwinia chrysanthemi38. In E. chrysanthemi, genes encoding enzymes responsible for pectin degradation and metabolism are located in close physical proximity, and the transfer of this genomic segment conferred E. coli with the ability to grow on di- and trigalacturonic acids. Construction (Steps 4–35). Although any plasmid can be used for system construction, single-copy plasmids or BACs (such as pBeloBAC11; Table 2) are recommended at this step, as they are able to maintain large DNA fragments at copy numbers that are representative of genome-based expression. Several methods currently exist for BAC manipulation, including direct cloning, recombineering and gene and parts assembly. In this protocol,

protocol Table 2 | Plasmids and antibiotic concentrations.

© 2014 Nature America, Inc. All rights reserved.

Plasmid

Antibiotics

Final concentration (mg/ml)

Function

pKm(R6Kγ)

Kanamycin

50

Template for overlap PCR and cassette construction

pCm(R6Kγ)

Chloramphenicol

50

Template for overlap PCR and cassette construction

pKD13

Ampicillin, kanamycin

100, 50

Template for overlap PCR and cassette construction

pDK46

Ampicillin

100

Expression of λ-RED recombinase system for recombineering

pCP20

Ampicillin

100

Expression of FLP recombinase for excision of FRT-flanked markers

pBeloBAC11

Chloramphenicol

25

BAC backbone

pJW168

Ampicillin

100

Expression of Cre recombinase for integration of lox-flanked cassettes

we focus on the construction of complex biological systems through overlap PCR cloning to build individual genetic modules followed by recombineering-based inchworm extension techniques for multimodule assembly on a BAC (Fig. 2). To construct each functional module, genetic components derived from various sources are first assembled together by overlap PCR with linearized pKm or pCm plasmids (Table 2). The pKm and pCm plasmids contain three basic components: the R6Kγ origin of replication, an antibiotic selection marker (for kanamycin and chloramphenicol resistance, respectively) and a 300-bp-long region with homology to the BAC to facilitate downstream recombineering. The spliced linear pKm- or pCm-based plasmid is circularized by treatment with T4 polynucleotide kinase and T4 DNA ligase and is subsequently transformed and maintained in E. coli (Fig. 2a). To construct the full biological system, each functional module is amplified from the pKm or pCm plasmid to yield an integration cassette flanked by a 300-bp sequence (derived from the pKm or pCm vector) and a 40-bp sequence (introduced by primers) with homology to the BAC backbone. Each module is then sequentially integrated into the BAC through λ-RED– mediated homologous recombination with the help of alternating antibiotic selection markers with the helper plasmid pKD46 (Figs. 2b and 3a and Table 2). Alternatively, a plasmid containing a flippase recognition target (FRT)-flanked selection marker such as pKD13 (ref. 32) can be used during overlap PCR assembly to allow for FLP recombinase-mediated marker recycling after each round of recombineering (Fig. 3b and Table 2). In V. splendidus 12B01, all genes thought to be necessary for alginate degradation and metabolism were located in close physical proximity to each other and were simultaneously cloned into the BACs with their native promoters and terminators (pALG3 and pALG7; pALG7 contains additional genomic segments from other organisms, see details in Supplementary Figs. 1 and 2). Implementation (Steps 36–54). Although RAGE can be conducted with any site-specific recombinase, this protocol focuses on the use of the Cre recombinase and mutually exclusive lox sites (e.g., a combination of the wild-type loxP and a mutant variant lox5171) to mediate the integration of the designed biological system into the bacterial chromosome (implementation)39–43 (Fig. 4). We begin by integrating a small targeting cassette

comprising a lox site–flanked selection marker (e.g., a chloramphenicol resistance gene) into the host genome at the desired integration locus by using standard recombineering protocols27,32. Next, this recipient strain is sequentially transformed with two constructs: (i) a vector expressing the Cre recombinase under the control of an inducible promoter (e.g., pJW168 from Lucigen, which has the Cre recombinase under the control of the IPTG-inducible lac promoter, Table 2) and (ii) the single-copy plasmid or BAC containing an integration cassette comprising the designed biological system and an FRT site–flanked selection marker (e.g., a kanamycin resistance gene, a different marker from the one present in the targeting cassette on the BAC). After these transformations are performed, Cre recombinase expression is induced and correct cassette integrations are selected by plating on kanamycin plates and checking for loss of chloramphenicol resistance. On the basis of our previous studies, we observe 100% sequence integrity after this simple antibiotic selection. After additional validation by PCR, sequencing or functional testing, the kanamycin selection marker is excised with the FLP recombinase32 to generate a markerless strain capable of undergoing additional rounds of genetic modification. Although the ‘plasmid delivery’ method is the recommended procedure for integration of single-copy plasmid– or BAC-based integration cassettes, there may be unique instances in which integration cassettes reside on other donor material such as multicopy plasmids or genomic DNA. In these cases, a ‘phage delivery’ method can be used for the integration of cassettes less than 90 kb long (Fig. 4). P1vir phage lysates are prepared from the donor strain containing the integration cassette and are used to infect recipient strains, which have been induced for Cre recombinase expression. Selection and validation of the integration then proceeds as described above. If the cassette is more than 90 kb, we suggest cloning into a BAC before proceeding with integration. Because of the ease of the implementation process, it is possible to simultaneously search across several parameters to quickly and efficiently optimize a biological system (Fig. 1). Different genetic host backgrounds, integration loci and intermodule configurations can be constructed in parallel and evaluated for the pheno­type of interest. Multiple copies of a given module can also be integrated through phage transduction (for systems less nature protocols | VOL.9 NO.6 | 2014 | 1323

a

Microbe A

Microbe B

A-F

AB-F

BC-F

B

C

A AB-R

Microbe C

a

(1)

A B C

(2)

A B C

D E F

(3)

A B C

D E F

G

(4)

A B C

D E F

G

b (1)

A B C

(2)

A B C

D E F

(3)

A B C

D E F

(4)

A B C

D E F

G

(5)

A B C

D E F

G

(6)

A B C

D E F

G

H

I

(7)

A B C

D E F

G

H

I

D E F

G

H

H

I

I

D E F

G

1324 | VOL.9 NO.6 | 2014 | nature protocols

H

I

R6Kγ

BC-R CpKm-R pKm-R (1) PCR amplification of each DNA fragment R6Kγ

B A

C (2) Overlap PCR to create a linear plasmid

A

B

R6Kγ

C

(3) Phosphorylation at 5′-OH with T4 PNK P

A

B

R6Kγ

C

P

(4) Ligation with T4 DNA ligase Kγ

A

R6

b

pKmD-F Forward primer

R6Kγ

(1) Amplification of integration cassette

D E F Reverse primer

BAC-based plasmid (pBACA-C) A B C

D E F OriS

repE parA parB parC

BAC-based plasmid (pBACA-F)

than 90 kb) or by repeating the RAGE protocol at different integration loci with additional pairs of mutually exclusive (i.e., noninteracting) lox sites. If the designed biological system does not perform as expected, it is possible to pursue additional iterations of design, construction and implementation to improve the system. This could include redesigning the biological system by considering other enzyme variants with enhanced activity or expression, adding

CpKm-F

C

Figure 2 | Module construction and recombineering into BACs. (a) Construction of individual modules/integration cassettes by overlap PCR cloning. (1) Amplify individual components for each module and a linear pKm, pCm or pKD13 plasmid from various sources (i.e., genomic, plasmid or synthetic DNA). Here pKm is shown as an example where the gray arrow denotes the kanamycin antibiotic-resistance marker. Primers used to splice two components consist of 50 nt of ss-oligo DNA, 25 bases of which are complementary to the first DNA fragment and the other 25 bases of which are complementary to the second DNA fragment. For example, AB-F denotes a primer used to amplify fragment B as a forward primer and to splice fragments A and B. (2) Splice together the individual components by overlap PCR by using standard PCR protocols. (3) Phosphorylate the 5′-OH groups of the assembled module (or integration cassette, now present as a linear form of a pKm-, pCm- or pKD13-based plasmid) with T4 polynucleotide kinase (T4 PNK). (4) Circularize this linear DNA fragment by T4 DNA ligase and transform it into an E. coli strain with a pir+ genotype for maintenance of R6Kγ ori-containing plasmids. (b) Manipulation of BACs through recombineering. Integration of a PCR-amplified DNA cassette (DEF-Km) from the plasmid constructed in a into a BAC-based plasmid (pBACA-C) is shown. (1) Amplify the integration cassette by PCR. The forward primers used for recombineering comprise 60 nt ss-oligo DNA, 20 bases of which are complementary to a sequence in the integration cassette (orange region in the forward primer), and the other 40 nt of which are complementary to the target sequence in the BAC-based plasmid (dark green region in the forward primer). The reverse primers comprise 25 nt of ss-oligo DNA (brown region in the reverse primer), which is complementary to the promoter region of the drug-resistant marker. The pKm-, pCm- and BAC-based plasmids are designed to share a 300-bp-long identical promoter sequence to improve the recombineering efficiency. (2) Recombination is catalyzed through expression of the λ-RED system on pKD46. Two identical sequences in the integration cassette and BAC (dark green: 40 bp long and brown: 300 bp long) are recombined with each other. The Cm selection marker is replaced with the Km selection marker during recombination.

B

© 2014 Nature America, Inc. All rights reserved.

protocol

A B C

D E F

(2) Integration via recombineering

OriS repE parA parB parC

novel functionalities or incorporating genetic elements for tuning pathway expression. The resulting genetically stable strain can also be further optimized through directed or adaptive evolutionary processes, such as multiplex automated genome engineering (MAGE)28. Figure 3 | Two methods for inchworm extension of BACs through recombineering. (a) Alternating selection markers (gray and white arrows designate the kanamycin- and chloramphenicol-resistance markers, respectively) can be used for the construction of large plasmids. (1) Each module (integration cassettes D–F, G, H and I) of the pKm- and pCm-based plasmids is amplified by PCR. (2) The first component (integration cassette) is integrated into the plasmid or BAC backbone (represented by fragments A–C) used for system construction and assembly. The kanamycin-resistance marker is replaced with the chloramphenicol-resistance marker during this process. (3) The same procedure is carried out with (1) and (2) to integrate a new module into the plasmid backbone. The chloramphenicolresistance marker is replaced with the kanamycin-resistance marker during this process. (4) Additional rounds of (1) and (2) can be repeated until the full biological system has been assembled. (b) A single drug-selection marker flanked by FRT sites (black bars) can be used for the construction of large plasmids. (1) Each module (integration cassettes as in (a)) of the pKD13-based plasmids is amplified by PCR. (2) The first module (integration cassette) is integrated into the plasmid or BAC backbone (represented as in a) used for system construction and assembly. (3) The kanamycin-resistance marker is excised through expression of a FLP recombinase. (4–7): steps (1–3) are repeated iteratively to integrate additional modules into the plasmid backbone until the full biological system has been assembled.

protocol a

b

Plasmid delivery

(1) Integrate the targeting cassette into the recipient strain via recombineering

Phage delivery

(1) Integrate the targeting cassette into the recipient strain via recombineering

Recipient strain +Arabinose

Recipient strain +Arabinose

loxP cat lox5171 loxP cat lox5171

pKD46 Chromosome

loxP cat lox5171

pKD46 Chromosome

Chromosome

(2) Transform the recipient strain with BAC containing the GOI and pJW168 Recipient strain

loxP cat lox5171

Chromosome

(2) Transform the recipient strain with pJW168 Recipient strain

pJW168

pJW168

BAC

BAC

GOI kan

GOI kan loxP cat lox5171

loxP cat lox5171

pJW168 loxP cat lox5171 Chromosome

pJW168 Chromosome

loxP cat lox5171 Chromosome

Chromosome

(3) Induce Cre recombinase expression

(3) Prepare phage lysates on a donor strain carrying the BAC containing the genes of interest (GOI) and infect a recipient strain induced for Cre recombinase expression

BAC +IPTG

P1vir

GOI kan

© 2014 Nature America, Inc. All rights reserved.

Donor strain

Recipient strain

BAC

GOI kan

pJW168

+IPTG

GOI kan

loxP cat lox5171 Chromosome

Chromosome

Chromosome

Chromosome

(4) Select kanamycin-resistant and chloramphenicol-sensitive colonies Kan

+IPTG

loxP cat lox5171

pJW168

lox5171

loxP

GOI kan

pJW168

GOI kan cat Chromosome

Chromosome

Cm

(4) Select kanamycin-resistant and chloramphenicol-sensitive colonies Kan

Figure 4 | RAGE is a genome engineering strategy that facilitates the stable integration of complex biological systems into the bacterial chromosome by recombinase-mediated cassette exchange. Genetically encoded biological systems Cm can be delivered into the cell in two ways. (a) Plasmid delivery. (1) Incorporate the chloramphenicol resistance gene (cat) flanked by two mutually exclusive lox sites (comprising the targeting cassette) into the bacterial genome through arabinose induction of the λ-RED recombination genes (encoded on pKD46). (2) Transform this recipient strain with both pJW168 (encoding the Cre recombinase) and the donor plasmid or BAC carrying the genes of interest (GOI) flanked by the same pair of lox sites. (3) Grow cells at 30 °C and induce Cre recombinase expression with IPTG. (4) Select correct recombinants on kanamycin plates and check them for chloramphenicol sensitivity. (b) Phage delivery. (1) Incorporate the chloramphenicol resistance gene (cat) flanked by two mutually exclusive lox sites (comprising the targeting cassette) into the bacterial genome through arabinose induction of the λ-RED recombination genes (encoded on pKD46). (2) Transform this recipient strain with pJW168. (3) Prepare P1vir lysates from a donor strain containing the GOI flanked by lox sites (either on a BAC, multicopy plasmid, or within the genome) and subsequently use it to infect a recipient strain induced for Cre recombinase expression. (4) Select correct recombinants on kanamycin plates and check them for chloramphenicol sensitivity.

MATERIALS REAGENTS • E. coli strains: DH5α (Life Technologies, cat. no. 18258-012); DH10B (Life Technologies, cat. no. 18290-015); ATCC8739 (American Type Culture Collection, cat. no. 8739); TransforMax EC100D pir+ Electrocompetent E. coli (Epicentre, cat. no. ECP09500) • Plasmids: pKm (Y. Yoshikuni, available from the author upon request), pCm (Y. Yoshikuni, available from the author upon request), pKD13 (no. 7633, Yale University, Coli Genetic Stock Center)32, pDK46 (no. 7669, Yale University, Coli Genetic Stock Center)32, pCP20 (no. 7629, Yale University, Coli Genetic Stock Center)32, pBeloBAC11 (New England Biolabs, cat. no. E4154S), pJW168 (Lucigen, cat. no. 42200-1) • Ampicillin (Sigma-Aldrich, cat. no. A0166) • Kanamycin (Sigma-Aldrich, cat. no. K0254) • Chloramphenicol (Sigma-Aldrich, cat. no. C7795) • Isopropyl β-d-1-thiogalactopyranoside (Sigma-Aldrich, cat. no. I5502) • Ethanol (Sigma-Aldrich, cat. no. E7023) • Agarose for molecular biology (Sigma-Aldrich, cat. no. 05066)

• Luria-Bertani (LB) broth, Miller (BD Difco, cat. no. 244610) • LB agar, Miller (BD Difco, cat. no. 244510) • Terrific broth, modified (TB; Sigma-Aldrich, cat. no. T0918) • SOC medium (New England Biolabs, cat. no. B9020S) • QIAprep miniprep kit (Qiagen, cat. no. 27106) • QIAquick PCR purification kit (Qiagen, cat. no. 28106) • QIAquick gel extraction kit (Qiagen, cat. no. 28706) • DNeasy blood and tissue kit (Qiagen, cat. no. 69504) • Phusion high-fidelity DNA polymerase (New England Biolabs, cat. no. M0530L) • Crimson Taq DNA polymerase (New England Biolabs, cat. no. M0324L) • T4 DNA ligase (New England Biolabs, cat. no. M0202M) • T4 polynucleotide kinase (New England Biolabs, cat. no. M0201L) • Restriction enzymes (New England Biolabs) • dNTPs, 100 mM (Life Technologies, cat. no. 10297-018) • SYBR Safe DNA gel stain (Life Technologies, cat. no. S33102) • Primers for PCR amplification, 25 pmol/ml in H2O (Integrated DNA Technologies) nature protocols | VOL.9 NO.6 | 2014 | 1325

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protocol • 50× TAE buffer (VWR International, cat. no. 97063-692) • Sterile distilled H2O • Nuclease-free water (Life Technologies, cat. no. AM9937) • Glycerol (Sigma-Aldrich, cat. no. G5516) • Magnesium chloride hexahydrate (MgCl2·6H2O; Sigma-Aldrich, cat. no. M2670) • Magnesium sulfate heptahydrate (MgSO4·7H2O; Sigma-Aldrich, cat. no. M5921) • Calcium chloride dihydrate (CaCl2·2H2O; Sigma-Aldrich, cat. no. C5080) • d-Dextrose (glucose) (Sigma-Aldrich, cat. no. G8270) • d-Arabinose (Sigma-Aldrich, cat. no. 10850) • Chloroform (Sigma-Aldrich, cat. no. C2432) ! CAUTION It is a hazardous chemical. Refer to the MSDS before use. • Sodium citrate monobasic (Sigma-Aldrich, cat. no. 71497) • P1vir bacteriophage (no. 12133, Yale University, Coli Genetic Stock Center)32 EQUIPMENT • Incubation shaker (I26, Eppendorf) • Spectrophotometer (UV1800, Shimadzu) • Safe Imager 2.0 blue-light transilluminator (Life Technologies, cat. no. G6600) • Molecular Imager Gel Doc XR+ system with Image Lab software (Bio-Rad Laboratories, cat. no. 170-8195) • C1000 Touch thermal cycler with Dual 48/48 Fast Reaction Module (Bio-Rad Laboratories, cat. no. 185-1148) • MicroPulser electroporator (Bio-Rad Laboratories, cat. no. 165-2100) • Gene Pulser/MicroPulser cuvettes (Bio-Rad Laboratories, cat. no. 165-2083) • PowerPac basic power supply (Bio-Rad Laboratories, cat. no. 164-5050) • Owl EasyCast B1 mini gel electrophoresis systems (B1, Thermo Fisher Scientific) • Cuvettes (VWR International, cat. no. 97000-586) • accuSpin Micro 17R microcentrifuge (Thermo Fisher Scientific, cat. no. 13-100-676) • Culture tubes, 14 ml (VWR International, cat. no. 60818-725) • Microcentrifuge tubes, 1.7 ml (VWR International, cat. no. 87003-294) • PCR tube strips, 0.2 ml (Thermo Fisher Scientific, cat. no. 14230215)

• Glass vials (VWR International, cat. no. 66011-041) • Axygen microtube racks (Thermo Fisher Scientific, cat. no. 14-222) • Petri dishes, 100 × 15 mm (VWR International, cat. no. 25384-302) • Syringes, 60 ml (VWR International, cat. no. 82002-318) • Syringe filters, 0.22 µm (VWR International, cat. no. 28200-018) • Sterile inoculating loops (VWR International, cat. no. 12000-810) • Autoclave (Yamato Scientific, cat. no. SM500) REAGENT SETUP LB medium  Prepare LB medium according to the vendor’s instructions. Add appropriate antibiotics to the medium at the concentrations specified in Table 2 before culturing E. coli. LB medium (without antibiotics) can be stored at room temperature (20–25 °C) for several months. LB agar  Prepare LB agar according to the vendor’s instructions. Autoclave the medium, cool it to ~50 °C and add appropriate antibiotics at the concentrations specified in Table 2. Pour the medium into Petri dishes to prepare solid medium. LB agar plates can be stored at 4 °C for up to 3 months. TB medium  Prepare TB medium according to the vendor’s instructions. Add appropriate antibiotics to the medium at the concentrations specified in Table 2 before culturing E. coli. TB medium (without antibiotics) can be stored at room temperature for several months. 10% (vol/vol) glycerol  Mix 100 ml of glycerol and 900 ml of distilled H2O. Autoclave to sterilize the medium and chill it to 4 °C before use. This solution can be stored at room temperature for several months. 100 mM CaCl2  Dissolve 0.735 g of CaCl2·2H2O in 50 ml of distilled H2O. Filter-sterilize the solution by passing it through a 0.22-µm syringe filter. This solution can be stored at room temperature for several months. 1 M MgCl2  Dissolve 10.17 g of MgCl2·6H2O in 50 ml of distilled H2O. Filter-sterilize the solution by passing it through a 0.22-µm syringe filter. This solution can be stored at room temperature for several months. 1 M MgSO4  Dissolve 12.32 g of MgSO4·7H2O in 50 ml of distilled H2O. Filter-sterilize the solution by passing it through a 0.22-µm syringe filter. This solution can be stored at room temperature for several months. 1 M sodium citrate  Dissolve 10.71 g of sodium citrate in 50 ml of distilled H2O. Filter-sterilize the solution by passing it through a 0.22-µm syringe filter. This solution can be stored at room temperature for several months.

PROCEDURE Designing a biological system ● TIMING 1–3 d 1| Design a biological system for a desired function and break it down into several smaller functional modules. Because design strategies will differ from pathway to pathway, it is difficult to generalize this procedure. Please see the Experimental design section for a summary of how we designed an E. coli platform capable of alginate degradation and metabolism25,26.  CRITICAL STEP When possible, define module boundaries for which inputs are readily available (e.g., sugar and metabolic intermediate substrates from commercial sources) and for which an output can be easily measured by conventional methods (e.g., cell growth, colorimetric and fluorescence assays, HPLC and GC). See examples in Table 1. 2| Design all necessary DNA sequences for promoters, genes and transcription terminators to build each functional module by using public databases, literature and web tools. ExPASy (http://www.expasy.org/resources) and the registry of standard biological parts (http://parts.igem.org/Main_Page) list many important databases and web tools. Software tools for in silico manipulation of plasmids will greatly improve the accuracy of construct design (e.g., Vector NTI, http://www.lifetechnologies.com/us/en/home/life-science/cloning/vector-nti-software.html). 3| On the basis of the in silico design, generate single-stranded (ss)-oligo DNA primers for PCR, overlap PCR, recombineering and sequencing. Rules for designing ss-oligo DNA primers for overlap PCR, recombineering and RAGE are described in Steps 4, 24, 36 and 40. Preparation of the integration cassette components ● TIMING 3–4 h 4| Assemble the following PCR reaction for each of the different integration cassette components and the linear pKm, pCm or pKD13 plasmids. Primers for splicing together two components should consist of 50 nt of ss-oligo DNA, 25 nt of which is

1326 | VOL.9 NO.6 | 2014 | nature protocols

protocol complementary to the first DNA fragment and the other 25 nt of which is complementary to the second DNA fragment. For a typical 50-µl PCR, assemble the following in a 0.2-ml PCR tube and mix the reaction solution well by pipetting: Component

Volume (ml)

© 2014 Nature America, Inc. All rights reserved.

5× Phusion high-fidelity buffer

Final concentration

10



DMSO (supplied with polymerase)

2.5

5%

10 mM dNTP

1

200 µM

10 µM forward primer

1

0.2 µM

10 µM reverse primer

1

0.2 µM

Phusion high-fidelity DNA polymerase

0.5

Template DNA (10 µg/ml)

1

0.2 µg/ml

Nuclease-free water

33



Total

50

1U

5| Perform the PCR with the following conditions: Cycle number

Denature

1

98 °C, 30 s

2–31

98 °C, 10 s

Anneal

Extend

60 °C, 15 s

72 °C, 30 s/kb

32

Hold

72 °C, 5 min

33

4 °C

6| Run the PCR products on an agarose gel, confirm the size of the amplified DNA fragments and gel-purify the appropriate bands by using the gel purification kit according to the manufacturer’s instructions. Elute the DNA into 30 µl of EB buffer (included in the kit). DNA concentration should be at least 10 µg/ml. Preparation of the integration cassette plasmid ● TIMING 3 d 7| To synthesize the DNA integration cassette (as a linear form of a pKm-, pCm- or pKD13-based plasmid) by overlap PCR, assemble the following 50-µl PCR reaction in a 0.2-ml PCR tube and mix the reaction solution well by pipetting: Component

Volume (ml)

Final concentration

5× Phusion high-fidelity buffer

10



DMSO (supplied with polymerase)

2.5

5%

10 mM dNTP

1

200 µM

10 µM forward primer

1

0.2 µM

10 µM reverse primer

1

0.2 µM

0.5

1U

5 each

Variable

up to 50



Phusion high-fidelity DNA polymerase Template DNA fragments prepared in Step 5 (cassette components and linear plasmids) Nuclease-free water Total

50

nature protocols | VOL.9 NO.6 | 2014 | 1327

protocol 8| Perform the PCR under the conditions described in Step 5.  CRITICAL STEP Although some protocols recommend increasing the extension time for each cycle, we have found that this standard PCR program often works better. ? TROUBLESHOOTING 9| Run the PCR product on an agarose gel, confirm the size of the amplified DNA and gel-purify the appropriate band by using the gel purification kit according to the manufacturer’s instructions. Elute the DNA into 30 µl of EB buffer. DNA concentration should be at least 10 µg/ml. 10| To circularize the integration cassette (which is now part of a linear pKm-, pCm or pKD13-based plasmid), treat it with T4 DNA polynucleotide kinase by incubating at 37 °C for 30 min. For a 9-µl reaction, mix the following in a 0.2-ml tube: Component

© 2014 Nature America, Inc. All rights reserved.

Solution prepared in Step 9

Volume (ml) 8

T4 DNA ligase buffer

0.5

T4 polynucleotide kinase

0.5

Total

9

11| Ligate the plasmid with T4 DNA ligase by incubating at 16 °C for 1 h. For a 10.5-µl reaction, mix the following in a 0.2-ml tube: Component

Volume (ml)

Solution prepared in Step 10

9

T4 DNA ligase buffer

1

T4 DNA ligase

0.5

Total

10.5

12| Mix 1 µl of the reaction solution with 40 µl of electrocompetent E. coli cells (TransforMax EC100D pir+), transfer the mixture into cuvettes and electroporate with 1.80 kV, 25 µF capacitance and 200 Ω resistance.  CRITICAL STEP Use an E. coli pir+ host strain for maintenance of plasmids with the R6Kγ origin of replication, such as TransforMax EC100D pir+. 13| Mix the transformants with 500 µl of SOC medium and incubate the mixture at 37 °C for 1 h, with shaking (200 r.p.m.). 14| Centrifuge the culture for 1 min at 17,000g at room temperature. Discard 500 µl of the supernatant and resuspend the cell pellet in the leftover supernatant. 15| Spread the resuspended cells on an LB agar plate containing the appropriate antibiotics and incubate the plate at 37 °C overnight. ? TROUBLESHOOTING 16| Inoculate 3–5 colonies separately into 5 ml of LB medium containing the appropriate antibiotics and grow them overnight at 37 °C with shaking (200 r.p.m.). 17| Collect the culture and isolate the plasmid by using the Qiagen miniprep kit according to the manufacturer’s instructions.  CRITICAL STEP Confirm correct inserts by restriction digestion and sequencing. Recombineering of integration cassettes into the BAC ● TIMING 3–5 d 18| Transform the λ-RED recombinase helper plasmid pKD46 into the desired host strain for BAC modification by using the heat shock transformation protocol described in Box 1. 19| Plate 100 µl of cells on an LB agar plate containing ampicillin and incubate the plate overnight at 30 °C.  CRITICAL STEP pKD46 contains a temperature-sensitive origin of replication. Strains containing pKD46 must be cultivated at 30 °C to prevent plasmid loss at higher temperatures. 1328 | VOL.9 NO.6 | 2014 | nature protocols

protocol Box 1 | Introduction of circular plasmids by heat-shock transformation ● TIMING 4–5 h  CRITICAL Owing to the relatively lower efficiency of heat-shock transformation, this procedure should only be used for the introduction of small (10–15 kb) are fragile. Avoid or minimize any mechanical impact that can break down the plasmids, including pipetting, vortexing, shaking and freezing. The use of wide-bore pipette tips can help minimize plasmid degradation. 35| Repeat Steps 4–34 for iterative rounds of inchworm extension. Integration of the targeting cassette by recombineering into host genome ● TIMING 2–3 d 36| Design primers for amplifying the targeting cassette that incorporate lox site sequences and at least 20–25 bp of homology to the desired integration locus (bold indicates the lox5171 site, underline indicates the loxP site). Gene amplified Cat

Template

Primer name sequence

Annealing temperature (°C)

Amplicon size (bp)

pCm

5′-(20–25 bp homology) ATAACTTCGTATAGTAC ACATTATACGAAGTTAT TCGGCACGTAAGAGGTTCCA ACTTT-3′

55

860

5′-(20–25 bp homology) ATAACTTCGTATAATGTAT GCTATACGAAGTTAT GGCGTTTAAGGGCACCAATAAC TGC-3′

55

1330 | VOL.9 NO.6 | 2014 | nature protocols

protocol Box 3 | Colony PCR ● TIMING 2–3 h 1. Design primers that span across the BAC and integration cassette junction with one primer binding in the integration cassette and one primer binding on the BAC or genome outside the flanking regions. 2. Assemble the following PCR reaction for verifying colonies. For multiple PCR samples, prepare a master mix and dispense 25-µl aliquots into 0.2-ml PCR tubes. A typical 25-µl reaction will contain the following: Component Volume (ml) Final concentration 5× Crimson Taq reaction buffer 5 1× 10 mM dNTP 0.5 200 µM 10 µM forward primer 0.5 0.2 µM 10 µM reverse primer 0.5 0.2 µM Crimson Taq DNA polymerase 0.125 0.625 U Nuclease-free water 18.4 — Total 25

© 2014 Nature America, Inc. All rights reserved.

3. Lightly touch a fresh colony with a sterile inoculating loop or pipette tip, mix the cells into the PCR tube and streak the loop or tip onto a fresh LB plate containing the appropriate antibiotic(s). 4. Perform the PCR with the following conditions: Cycle number Denature Anneal Extend 1 95 °C, 5 min 2–31 95 °C, 30 s 55 °C, 30 s 68 °C, 1 min/kb 32 68 °C, 5 min 33

Hold

4 °C

5. Visualize the products on an agarose gel.

37| Assemble the following PCR reaction for amplifying the targeting cassette from plasmid pCm by using the primer pairs designed in Step 36. For a typical 50-µl PCR, assemble the following in a 0.2-ml PCR tube and mix the reaction solution well by pipetting: Component

Volume (ml)

Final concentration

5× Phusion high-fidelity buffer

10



DMSO (supplied with polymerase)

2.5

5%

10 mM dNTP

1

200 µM

10 µM forward primer

1

0.2 µM

10 µM reverse primer

1

0.2 µM

0.5

1U

1 pg–10 ng

Variable

Nuclease-free water

33



Total

50

Phusion high-fidelity DNA polymerase PCm

 CRITICAL STEP Make sure that two mutually exclusive lox sites (e.g., loxP and lox5171) are incorporated into the 5′ sequence of these primers.  CRITICAL STEP Amplify the selection marker from a plasmid containing the R6Kγ origin to prevent false positives due to transformation of contaminating circular plasmid in the PCR product (plasmids containing the R6Kγ origin cannot be replicated in normal E. coli host strains). Alternatively, non-R6Kγ plasmid templates can be linearized with a restriction enzyme, which is present in the vector backbone but absent in the selection marker, before use as a template for PCR amplification 27. 38| Perform the PCR under the conditions described in Step 5. 39| Run the PCR product on an agarose gel, confirm the size of the amplified DNA fragment, and gel-purify the appropriate band by using a gel purification kit (e.g., Qiagen). nature protocols | VOL.9 NO.6 | 2014 | 1331

protocol 40| Use the gel-purified product as a template for a second round of PCR amplification (by following Steps 37 and 38) with a set of primers that bind to the 20–25-bp homology region designed in Step 30 and, additionally, that extend the homology region to the site of integration.  CRITICAL STEP Between the primers used for the first and second rounds of PCR amplification, be sure to incorporate at least 50 bp of flanking homology at each end to the desired site of integration. Higher integration efficiencies can be obtained with longer homology regions. 41| Purify the PCR product with a PCR purification kit according to the manufacturer’s instructions, quantify the DNA concentration with a spectrophotometer (measure absorbance at 260 nm; A260) and visualize the product on an agarose gel.  CRITICAL STEP Elute the DNA in water instead of EB buffer to prevent arcing during electroporation. 42| Prepare λ-RED recombination–induced electrocompetent cells for your recipient strain as described in Box 2. 43| Mix at least ~250 ng of the purified PCR product with 50 µl of electrocompetent cells, transfer the mixture into cuvettes and electroporate with 1.80 kV, 25 µF capacitance and 200 Ω resistance.

© 2014 Nature America, Inc. All rights reserved.

44| Mix the transformants with 1 ml of SOC medium and incubate the mixture at 37 °C for 1 h, with shaking (200 r.p.m.). 45| Spread 200 µl of cells on LB agar–chloramphenicol plates and incubate them at 37 °C overnight. Leave the rest of the cells at room temperature overnight. These can be plated the following day if insufficient colonies are recovered from the original plate. 46| Confirm the accuracy of recombination by performing colony PCR (Box 3) across both integration junctions, with one primer binding in the selection cassette and one primer binding on the genome outside the flanking regions. Confirm the integrity of the lox sites by sequencing. ? TROUBLESHOOTING 47| Check for loss of pKD46 by taking toothpicks or pipette tips and streaking the colonies on two separate agar plates: LB agar + chloramphenicol and LB agar + ampicillin. Sensitivity to ampicillin indicates that the pKD46 plasmid has been properly cured. ? TROUBLESHOOTING Delivery and recombinase-mediated insertion of the integration cassette ● TIMING 4–7 d 48| Transform the Cre recombinase–expressing vector pJW168 into the targeting cassette–containing recipient strain by using the heat-shock transformation protocol described in Box 1. 49| Plate 100 µl of cells on LB plates containing ampicillin and incubate them overnight at 30 °C.  CRITICAL STEP pJW168 contains a temperature-sensitive origin of replication. Strains containing pJW168 must be cultivated at 30 °C to prevent plasmid loss at higher temperatures. 50| Introduce the integration cassette into a targeting cassette–containing recipient strain by using the plasmid-based delivery (option A; Fig. 4a) if the lox-flanked integration cassette is present within a BAC or single-copy plasmid; or by using phage-based delivery (option B; Fig. 4b) if the lox-flanked integration cassette is present within a BAC, single-copy plasmid, multicopy plasmid or bacterial genome (if this option is used, owing to limits on the size of DNA packaged by the P1 phage, the integration cassette must not exceed 90 kb): (A) Plasmid-based delivery and integration (i) Inoculate 5 ml of LB medium containing kanamycin with a colony (Step 35) or glycerol stock of a donor strain carrying a BAC or single-copy plasmid containing the lox-flanked integration cassette. (ii) Isolate the BAC or single-copy plasmid by using the Qiagen miniprep kit according to the manufacturer’s instructions and quantify the DNA concentration by using a spectrophotometer (A260).  CRITICAL STEP Long BAC-based plasmids (>10–15 kb) are fragile. Avoid or minimize any mechanical impact that can break down the plasmids, including pipetting, vortexing, shaking and freezing. The use of wide-bore pipette tips can help minimize BAC-based plasmid degradation. (iii) Prepare electrocompetent cells from a strain carrying pJW168 and the lox-flanked targeting cassette (Step 49) by following the procedure outlined in Box 2.  CRITICAL STEP pJW168 contains a temperature-sensitive origin of replication. Strains containing pJW168 must be cultivated at 30 °C to prevent plasmid loss at higher temperatures. (iv) Mix ~100 ng of the integration cassette–containing BAC or single-copy plasmid with 50 µl of electrocompetent cells, transfer the mixture into cuvettes and electroporate with 1.80 kV, 25 µF capacitance and 200 Ω resistance. 1332 | VOL.9 NO.6 | 2014 | nature protocols

protocol Box 4 | Deletion of selectable marker flanked by FRT sites ● TIMING 3 d

© 2014 Nature America, Inc. All rights reserved.

1. Transform the strain containing the FRT-flanked selectable marker with pCP20 (for FLP recombinase expression) by using the heat-shock transformation method described in Box 3. 2. Spread cells on LB-ampicillin agar plates and incubate them at 30 °C for maintenance of the temperature-sensitive plasmid pCP20. 3. The following day, streak colonies onto LB agar plates (without antibiotic) and incubate at 42 °C to promote plasmid loss. 4. The following day, streak individual colonies on LB agar plates, LB-ampicillin agar plates and LB-antibiotic agar plates (containing the antibiotic whose resistance gene was flanked by FRT sites), and incubate at 37 °C. Colonies with deleted antibiotic markers should not grow on LB-antibiotic agar plates (indicating marker excision) or on LB-ampicillin agar plates (indicating proper curing of pCP20). 5. Confirm the loss of the antibiotic marker by colony PCR (Box 2) and/or sequencing.

(v) Spread 100 µl of the cells on an LB agar plate containing ampicillin and kanamycin and incubate the plate at 30 °C overnight. (vi) Take a single colony and inoculate it into 5 ml of LB medium with ampicillin and kanamycin and incubate at 30 °C overnight. (vii) Transfer 25 µl of the overnight culture into 2.5 ml of fresh LB medium containing ampicillin, kanamycin and 1 mM IPTG and incubate it at 30 °C for at least 3 h. Longer incubation times do not affect the efficiency of recombination. (viii) Spot 10 µl of the culture onto an LB agar plate containing kanamycin and spread it with an inoculation loop to isolate single colonies. Incubate the plate at 37 °C overnight.  CRITICAL STEP Incubation is performed at 37 °C to promote the loss of the pJW168 plasmid. (B) Phage-based delivery and integration (i) Inoculate 5 ml of LB medium containing kanamycin from a colony (Step 35) or glycerol stock of a donor strain carrying the lox-flanked integration cassette on a BAC, single-copy plasmid, multicopy plasmid, or within its genome. Grow the culture overnight at 37 °C. (ii) Transfer 30 µl of the overnight culture into 3 ml of fresh LB medium supplemented with 25 mM MgCl2, 5 mM CaCl2 and 0.1% (wt/vol) glucose. (iii) Incubate the culture at 37 °C for 1–2 h (until there is noticeable growth); add 40 µl of P1vir phage lysate and continue incubating at 37 °C for 1–3 h until the culture has completely lysed. ? TROUBLESHOOTING (iv) Add 100 µl of chloroform to the lysate; vortex and centrifuge at 4,000g for 5 min at room temperature. ! CAUTION Chloroform is a hazardous chemical. Refer to the MSDS before use. (v) Transfer the supernatant into a glass vial and add 50–100 µl of chloroform.  PAUSE POINT Phage lysates can be stored at 4 °C for several months until needed. (vi) Inoculate 2 ml of LB medium containing ampicillin and 1 mM of IPTG with a colony (Step 49) or glycerol stock of a recipient strain carrying pJW168 and the lox-flanked targeting cassette. Grow the culture overnight at 30 °C. (vii) Centrifuge the culture at 4,000g for 5 min at room temperature, discard the supernatant and resuspend the cells in 600 µl of LB medium containing 100 mM MgSO4 and 5 mM CaCl2. (viii) Set up the following four separate transduction reactions in 14-ml culture tubes and incubate them at 37 °C (without shaking) for 30 min. Reaction 1: 100 µl of undiluted P1 phage lysate (Step 50B(v)) + 100 µl of recipient cells; reaction 2: 100 µl of a 1:10 diluted P1 phage lysate (in LB medium) + 100 µl of recipient cells; reaction 3, 100 µl of LB + 100 µl of recipient cells; and reaction 4: 100 µl of undiluted P1 phage lysate + 100 µl of LB.  CRITICAL STEP Reactions 1 and 2 are transduction reactions performed with different amounts of phage. If colonies are obtained from both reactions, it is best to pick colonies from the reaction that uses the least amount of phage (reaction 2). Reaction 3 is a negative control to ensure that the recipient cells are not resistant to your antibiotic selection. Reaction 4 is a negative control to ensure that no live donor cells are present in the phage lysate. Reactions 3 and 4 should not yield any colonies.  CRITICAL STEP Use LB medium containing 100 mM of MgSO4 and 5 mM of CaCl2.  CRITICAL STEP When setting up the infection reactions, tap the phage lysate vials to allow the chloroform to settle to the bottom before pipetting, and be sure not to pipette from this chloroform layer. (ix) Add 200 µl of 1 M sodium citrate and 1 ml of LB medium (without MgSO4 or CaCl2) to each tube and incubate at 37 °C. (x) Centrifuge the cultures at 4,000g for 5 min at room temperature, discard the supernatant and resuspend the cells in 100 µl of LB medium with 100 mM sodium citrate. (xi) Plate cells on LB plates containing kanamycin and incubate at 37 °C overnight.  CRITICAL STEP Incubation is performed at 37 °C to promote loss of the pJW168 plasmid. ? TROUBLESHOOTING nature protocols | VOL.9 NO.6 | 2014 | 1333

protocol 51| Screen ten colonies by taking a toothpick or pipette tip and streaking each colony on three separate plates: LB agar + chloramphenicol, LB agar + kanamycin, and LB agar + ampicillin. Correct recombinants should be resistant to kanamycin and sensitive to chloramphenicol. Sensitivity to ampicillin indicates that the pJW168 plasmid has been properly cured. ? TROUBLESHOOTING 52| Confirm the accuracy of recombination by performing colony PCR (Box 3) across both integration junctions with one primer binding in the selection cassette and one primer binding on the genome outside the flanking regions. Integrity of the cassette can also be verified by sequencing or functional testing. 53| For colonies derived from phage-based delivery only, to remove phage contamination, use a toothpick or pipette tip to pick correct colonies and restreak them on fresh LB agar plates pre-spread with 100 µl of 1 M sodium citrate. Repeat this step if necessary until the colony morphology is round, indicating absence of phage infection.  CRITICAL STEP Make sure that the phage is completely removed at this point to prevent contamination at downstream steps.

© 2014 Nature America, Inc. All rights reserved.

54| (Optional) To allow for additional rounds of genetic manipulation, selectable markers can be recycled through FLP-mediated marker excision as described in Box 4. ? TROUBLESHOOTING Troubleshooting advice can be found in Table 3. Table 3 | Troubleshooting table. Step

Problem

Possible reason

Solution

8

No spliced PCR products

Inadequate splicing sites and primers

Redesign splicing sites and primers

Inadequate annealing temperature

Try different annealing temperatures

Inadequate PCR conditions

Try different Mg2+ concentration; use betaine (B0300, Sigma-Aldrich) or Q-solution (Qiagen) instead of DMSO

High GC content of template

Use Phusion GC buffer

Efficiency of electrocompetent cells is too low

Use electrocompetent cells with efficiencies >109–1010 c.f.u./µg DNA

Ligation efficiency is too low

Use new T4 DNA ligation buffer, T4 PNK, and T4 DNA ligase

The concentration of the integration cassette is too low

Increase the DNA concentration used for transformation

Efficiency of electrocompetent cells is too low

Use electrocompetent cells with efficiencies >109–1010 c.f.u./µg DNA

15

30, 46

No transformants are obtained

No transformants are obtained

31, 47

Colonies are resistant to ampicillin

Ineffective curing of plasmid pKD46

Streak out cells on fresh LB agar plates and grow at 42 °C to facilitate plasmid loss

50B(iii)

Culture did not lyse

Low titer of phage stock

Allow lysis to continue overnight. Culture will be completely saturated but should have noticeable cellular debris. Prepare phage lysate from this culture

50B(xi)

Colonies growing on negative control plate ‘3’

Recipient cells already contain the antibiotic marker being used for selection

Remove antibiotic marker from recipient cells or use another selection marker for the transduction

Contaminated LB medium

Use sterile LB medium

Colonies growing on the negative control plate ‘4’

Donor strain is still present in the phage lysate

Add an additional 100 µl of chloroform to the phage lysate and vortex completely to kill residual donor cells

Colonies are resistant to ampicillin

Ineffective curing of plasmid pJW168

Streak out cells on fresh LB agar plates and grow them at 42 °C to facilitate plasmid loss

51

1334 | VOL.9 NO.6 | 2014 | nature protocols

protocol

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● TIMING Steps 1–3: 1–3 d Steps 4–6: 3–4 h Steps 7–17: 3 d Steps 18–35: 3–5 d Steps 36–47: 2–3 d Steps 48–54: 4–7 d Box 1: 4–5 h Box 2: 4–5 h Box 3: 2–3 h Box 4: 3 d ANTICIPATED RESULTS As mentioned previously, the application of this strainengineering paradigm markedly expedited the construction Table 4 | Tested configurations for the construction of BAL1611. of an optimal E. coli–based platform for the production Tested configurations of biofuels and renewable chemicals from brown 25,26 macroalgae . The incorporation of two mutually Background strains MG1655, W3110, ATCC8739, BL21, DH5α exclusive lox site pairings (e.g., loxP, lox5171) flanking the constructed alginate utilization modules then Integration loci ldhA, frd, pfl-focB, int(gidB-atpI), facilitated the integration of these large genetic fragments int(mraZ-fruR) into precise and predetermined locations within the Copy number 1, 2 bacterial genome via RAGE. Notably, the simplicity and speed of this approach allowed for the parallelized implementation of three functional modules (alginate secretion machinery, the alginate uptake and metabolism pathway and the ethanol production pathway) to explore the effects of various configurations (e.g., genetic background, integration loci, copy number and intermodule interactions and compatibility as described in Table 4) on strain performance. These explorations yielded BAL1611, a strain showing optimal cellular phenotypes (high ethanol production yield, titer, and productivity from brown macroalgae) with demonstrated genetic stability over its plasmid-based counterpart over at least 50 generations.

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Engineering complex biological systems in bacteria through recombinase-assisted genome engineering.

Here we describe an advanced paradigm for the design, construction and stable implementation of complex biological systems in microbial organisms. Thi...
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