Key words

1 Introduction

Many cellular processes depend on multi-subunit protein complexes [1]. When individual subunits of a protein complex are coexpressed in heterologous cell systems, they often assemble into functional protein complexes that can be purified and then used for structural and functional studies. The baculovirus-insect cell system is an invaluable tool for the production of recombinant proteins and protein complexes that cannot be easily produced in E. coli , for example, because they might require chaperone and posttranslational modification systems provided by eukaryotic host cells. Typically, the coding sequences for proteins to be expressed are cloned into expression cassettes on a baculoviral transfer vector before these expression cassettes are transferred onto a baculoviral genome using Tn7 transposition [2]. The yield and homogeneity of protein complex preparations can be improved if all subunits are expressed from a single baculoviral vector rather than from combinations of single subunit baculoviral vectors [3]. Prominent transfer vectors are the pFastBac vectors onto which one or two genes can be cloned. For the coexpression of several subunits, the MultiBac series of transfer vectors are particularly useful tools that utilize a range of cloning techniques including conventional restriction-ligation cloning, sequence- and ligation-independent cloning (SLIC) combined with Cre-LoxP recombination of “acceptor” and “donor” vectors (tandem recombineering), and uracil-specific excision reagent (USER) cloning [4,5,6,7,8].

Here we describe a detailed protocol for the biGBac cloning method [9] that uses Gibson assembly reactions [10] to generate baculoviral transfer vectors coding for up to 25 subunits of a protein complex. The biGBac assembly procedure (Fig. 1) uses three assembly levels corresponding to three types of biGBac cloning vectors (Fig. 2).

Fig. 1
figure 1

biGBac assembly procedure. (a) Each cDNA is cloned into a gene expression cassette (GEC) in the pLIB vector. A GEC consists of a polyhedrin promoter (prom), the coding sequence of the protein to be expressed (cDNA), and a transcriptional terminator sequence (term). GECs are amplified by PCR from pLIB templates using predefined primer sets (Cas_for/Cas_rev primers; see Table 1) that introduce linker sequences for Gibson assembly (Greek letters). The “last” GEC of a pBIG1 assembly should carry the “omega” linker sequence (Casω_rev) to create an overlap with the pBIG1 vector. (b) In the first assembly step, up to five PCR-amplified GECs containing suitable linker sequences are combined in a Gibson assembly reaction with a linearized pBIG1 vector generating a polygene cassette (PGC). pBIG1 constructs are analyzed by SwaI digestion to release individual GECs or by PmeI digestion to release the PGC. After PmeI digestion the released PGC contains new linker sequences on the fragment ends (indicated as A, B, …). (c) In the second assembly step, up to five PGCs from different pBIG1 constructs are released by PmeI digestion and combined in a Gibson assembly reaction with a linearized pBIG2 vector. Generated pBIG2 constructs are analyzed by SwaI digestion to release individual GECs or by PacI digestion to release PGCs

Full size image
Fig. 2
figure 2

Schematic representation of biGBac cloning vectors. (a) pLIB vector level: the coding sequence of a subunit (cDNA) is cloned into the BamHI-/HindIII-linearized pLIB vector, which generates a gene expression cassette (GEC) consisting of polyhedrin promoter (prom), cDNA, and transcriptional terminator sequence (term). Generated pLIB constructs can be used as templates for GEC amplification and multigene construct generation in pBIG1 or for transfer of its GEC to a baculoviral genome using Tn7 transposition (Tn7R, Tn7L sites and gentamicin resistance). pLIB is maintained with ampicillin (AmpR). (b) pBIG1 vector level: amplified GECs from pLIB templates are inserted into SwaI-linearized pBIG1 vectors. All pBIG1 vectors contain the “alpha” and “omega” linker sequences for GEC insertion (α, ω) at the SwaI linearization site. PmeI digestion of pBIG1 constructs releases the generated polygene cassette (PGC), and new linker sequences (A, B, C, D, E, or F) appear on the fragment ends depending on which of the five pBIG1 vectors a, b, c, d, or e was used. Generated pBIG1 constructs can be used to generate larger multigene assemblies in pBIG2 vectors or to transfer its GECs onto a baculoviral genome using Tn7 transposition (Tn7R, Tn7L sites, and GentaR). Spectinomycin resistance (SpecR) is used as selection marker for pBIG1 construct generation. (c) PGCs from different pBIG1 constructs with compatible linker sequences are inserted into PmeI-linearized pBIG2 vectors. The name of the pBIG2 vector (ab, abc, …) indicates which pBIG1-derived PGCs can be combined. Generated pBIG2 constructs can be used to transfer its GECs onto a baculoviral genome using Tn7 transposition (Tn7R, Tn7L, and GentaR). Chloramphenicol resistance (CamR) is used as selection marker for pBIG2 construct generation

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On the first level, the cDNAs of interest are individually cloned into gene expression cassettes (GECs) on the transfer vector pLIB (library vector). The GEC consists of the subunit cDNA flanked by a polyhedrin promoter for high expression levels in baculovirus-infected insect cells and a transcriptional terminator sequence.

On the second level , the GECs are amplified by PCR from pLIB templates using predefined primer sets (Table 1) that introduce DNA linker sequences for Gibson assembly at the fragment ends (Fig. 1a). Up to five PCR-amplified GECs are combined in a Gibson assembly reaction with a linearized pBIG1 vector to create a transfer vector coding for up to five subunits. This procedure introduces SwaI restriction sites between individual GECs and PmeI sites flanking the generated polygene cassette (PGC) for convenient analysis of clones (Fig. 1b). Digestion by PmeI additionally leads to the appearance of new linker sequences on the fragment ends of the PGC. This multi-GEC assembly step can be performed in five different versions of the pBIG1 vector—distinguished by the letters a, b, c, d, and e—that differ only in the linker sequences next to the PmeI sites (see Fig. 2b).

Table 1 Predefined primer set for GEC amplification
Full size table

On the third level, up to five PGCs derived from different pBIG1 constructs can be combined after PmeI digestion with a compatible linearized pBIG2 vector (see Fig. 2c) in a Gibson assembly reaction to create a transfer vector that can encode up to 25 subunits. Products of this multi-PGC assembly step can be analyzed by SwaI digestion cleaving between individual GECs or by PacI digestion cleaving between PGCs (Fig. 1c).

All three types of biGBac vectors (pLIB, pBIG1, pBIG2) contain Tn7L and Tn7R sites as well as a gentamicin resistance marker, which can be used to generate recombinant baculoviral genomes using Tn7 transposition (Fig. 2). The linker sequences used in biGBac have been carefully selected and tested for high assembly efficiency and specificity in the Gibson assembly steps used in biGBac [9]. These linker sequences are encoded on the vector backbones and the predefined primer set.

The biGBac cloning method has been used successfully to reconstitute protein complexes such as the anaphase-promoting complex/cyclosome, mitotic checkpoint complex, cohesin , and kinetochore complexes [9, 11,12,13]. In principle, biGBac can be used for the coexpression of any proteins, not only for subunits of a protein complex, for example, to coexpress a chaperone to assist in protein folding or to coexpress a posttranslational modification enzyme with its substrate.

2 Materials

2.1 Generation of biGBac Multigene Transfer Vectors

  1. 1.

    biGBac vectors (Addgene kit #1000000088): pLIB, pBIG1a, pBIG1b, pBIG1c, pBIG1d, pBIG1e, pBIG2ab, pBIG2abc, pBIG2abcd, and pBIG2abcde.

  2. 2.

    2× Gibson assembly master mix (e.g., NEB) (see Note 1 ).

  3. 3.

    Restriction endonucleases: BamHI, HindIII, PacI, PmeI, and SwaI.

  4. 4.

    Standard PCR reagents and equipment: thermocycler and high-fidelity DNA polymerase (e.g., Phusion polymerase, Thermo Fisher Scientific).

  5. 5.

    Predefined DNA oligonucleotide set for GEC amplification (e.g., Microsynth; PAGE-purified quality recommended): five Cas_for and five Cas_rev primers (Table 1).

  6. 6.

    Standard agarose gel electrophoresis reagents and equipment.

  7. 7.

    Miniprep and gel extraction kits.

  8. 8.

    PureLink PCR Purification Kit (Thermo Fisher Scientific) .

  9. 9.

    Spectrophotometer.

  10. 10.

    Competent cells of a standard E. coli cloning strain (e.g., DH10B) (see Note 2 ).

  11. 11.

    Media and antibiotics for growing E. coli cultures: LB medium, LB-agar plates, ampicillin (use at 100 μg/ml), spectinomycin (use at 50 μg/ml), and chloramphenicol (use at 34 μg/ml).

  12. 12.

    Sequencing primers P1 (5′-TCAACAGGTTGAACTGCTGATC-3′) and P2 (5′-GGTGTAGCGTCGTAAGCTAATAC-3′) and gene-specific sequencing primers.

2.2 Generation of Recombinant Baculoviruses from biGBac Transfer Vectors Using Tn7 Transposition

  1. 1.

    E. coli competent cells for site-specific transposition onto baculoviral genome with Tn7 (e.g., DH10EMBacY [14]).

  2. 2.

    Blue-white selection plates: LB-agar, 50 μg/ml X-gal, 0.1 mM IPTG, 50 μg/ml kanamycin, 10 μg/ml tetracycline, and 7 μg/ml gentamicin.

  3. 3.

    Isopropanol and ethanol.

  4. 4.

    Sf9 insect cells and cell culture media (Thermo Fisher Scientific).

  5. 5.

    Transfection reagent (e.g., Fugene 6, Promega).

3 Methods

We recommend, in particular for large complexes, to plan how to distribute subunits between biGBac vectors. It is recommended to first generate linearized stocks of the biGBac cloning vectors (see Subheading 3.1) and to clone each subunit into the pLIB vector to generate a “library” of pLIB constructs, each containing one subunit. Any additional DNA sequences required, for example, those encoding affinity tags or protease recognition sites, should be introduced at this stage (see Subheading 3.2). In the first assembly step, up to five subunits can be combined on one pBIG1 vector (see Subheading 3.3). It can be useful to collect subcomplexes on pBIG1 vectors that might be expressed directly from pBIG1-derived baculoviruses or to put subunits that will be mutagenized onto a separate pBIG1 vector. Up to five PGCs from different pBIG1 constructs can be combined on a pBIG2 vector (see Subheading 3.4). To determine which pBIG1 vectors are compatible with which pBIG2 vectors and to plan how to distribute subunits, refer to Fig. 2. Another consideration is that pBIG2 assemblies are typically more efficient than pBIG1 assemblies, in particular if pBIG1 assemblies combine several large subunits. To reduce the number of clones that need to be analyzed, it can be useful to distribute the largest subunits onto several pBIG1 constructs and to reduce the number of subunits per pBIG1 construct.

3.1 Preparation of Linearized Cloning Vectors

  1. 1.

    Linearize the pLIB vector by digestion with BamHI/HindIII: mix 5 μl 10× FastDigest buffer, 30 μl pLIB plasmid DNA (~300 ng/μl), 2 μl BamHI, 2 μl HindIII, and 11 μl water, and incubate at 37 °C for 3 h. Gel-purify using a 0.7% agarose gel and a gel extraction kit.

  2. 2.

    Linearize the five pBIG1 vectors by digesting each pBIG1 vector with SwaI: mix 5 μl 10× NEBuffer 3.1, 30 μl pBIG1 plasmid DNA (~300 ng/μl), 1 μl SwaI, and 14 μl water, and incubate at 25 °C overnight. Add another 2 μl SwaI for 2 h. Heat-inactivate SwaI at 65 °C for 20 min and purify the linearized plasmid using a PCR purification kit (see Note 3 ).

  3. 3.

    Linearize the four pBIG2 vectors by digesting each pBIG2 vector with PmeI: mix 5 μl 10× CutSmart buffer, 30 μl pBIG2 plasmid DNA (~300 ng/μl), 1 μl PmeI, and 14 μl water, and incubate at 25 °C overnight. Add another 2 μl PmeI for 2 h. Purify the linearized plasmid using a PCR purification kit.

3.2 Cloning of pLIB Library Constructs

  1. 1.

    PCR-amplify the cDNA of a protein complex subunit using oligonucleotide primers containing the 5′ extensions 5′-CCACCATCGGGCGCGGATCCA... (followed by start-ATG and gene-specific sequences) for the forward primer and 5′-TCCTCTAGTACTTCTCGACAAGCTT... (followed by reverse complement of stop codon and gene-specific sequences) for the reverse primer. In case the cDNA sequence contains a PmeI recognition site (GTTTAAAC), amplify the cDNA as two overlapping PCR products choosing a Gibson overhang that introduces a silent mutation to the PmeI site (see Note 4 ). At this stage, tags can be introduced to subunits (see Note 5 ).

  2. 2.

    Purify the PCR products by gel extraction from a 0.7% (w/v) agarose gel using a gel extraction kit, elute DNA in 30 μl EB buffer and determine DNA concentration using a spectrophotometer.

  3. 3.

    Prepare the Gibson assembly reaction by mixing 100 ng BamHI-/HindIII-digested pLIB vector DNA (see Subheading 3.1, step 1) and a 5× molar excess of PCR-amplified cDNA in a volume of 5 μl (see Note 6 ). Add water to 10 μl total volume. Preheat a thermocycler block to 50 °C with heated lid.

  4. 4.

    Perform the Gibson assembly reaction by adding 10 μl 2× Gibson assembly master mix on ice and mix by pipetting up and down. Immediately (see Note 7 ) transfer the tube to the preheated 50 °C thermocycler and incubate for 1 h (see Note 8 ).

  5. 5.

    Mix the finished Gibson assembly reaction by flicking the tube and transform a standard E. coli cloning strain such as DH10B (see Note 2 ). Recover the transformed bacteria in LB medium at 37 °C for 60 min, spread on LB-ampicillin agar plates and incubate at 37 °C overnight.

  6. 6.

    Pick 2–6 colonies per pLIB construct and grow 5 ml cultures at 37 °C in LB-ampicillin medium overnight and isolate plasmid DNA using a miniprep kit.

  7. 7.

    Digest 1.5 μl of plasmid DNA (~80–500 ng/μl) with BamHI/HindIII: mix 1 μl 10× FastDigest buffer, 0.2 μl FastDigest BamHI, 0.2 μl FastDigest HindIII, 1.5 μl DNA, and 7.1 μl water, and incubate at 37 °C for 1–3 h, and analyze by electrophoresis on a 0.8% (w/v) agarose gel.

  8. 8.

    Sequence clones that show the expected restriction pattern by Sanger sequencing using sequencing primers P1 and P2 and gene-specific primers.

3.3 Cloning of pBIG1 Constructs Coding for Up to Five Subunits

  1. 1.

    Choose up to five pLIB constructs containing the cDNAs of subunits to be combined on a pBIG1 vector, and use them as templates in PCR reactions to amplify linker sequence-flanked GECs: mix 20 μl 5× HF buffer, 2 μl dNTPs (10 mM each), 0.5 μl CasX_for primer (100 μM) (see Table 1), 0.5 μl CasX_rev primer (100 μM) (see Table 1), 0.5 μl pLIB template, 1 μl Phusion HF polymerase, and 75.5 μl water. Use the Casω_rev primer for the “last” cassette to generate a Gibson overlap to the vector (see Fig. 1b). Set up the PCR reactions on ice and run the following PCR program: 95 °C for 3 min, 42× (98 °C for 15 s, 65 °C for 20 s, 72 °C for 30 s/kb), 72 °C for 5 min; store at 4 °C (see Note 9 ).

  2. 2.

    Purify the PCR-amplified GECs using the PureLink PCR Purification Kit . Mix 400 μl high-cutoff binding buffer B3 with 100 μl PCR product. Perform the purification according to the manufacturer’s instructions. Elute in 30 μl E1 buffer. Determine DNA concentration using a spectrophotometer (see Note 10 ). Confirm successful amplification and purity of the GECs by running 3 μl aliquots on a 0.8% (w/v) agarose gel.

  3. 3.

    Prepare a Gibson assembly reaction by mixing 100 ng SwaI-digested pBIG1 vector (see Subheading 3.1, step 2), a 5× molar excess of each PCR-amplified GEC, and water to a volume of 10 μl (see Note 11 ). Preheat a thermocycler block to 50 °C with heated lid.

  4. 4.

    Perform a Gibson assembly reaction by adding 10 μl 2× Gibson assembly master mix on ice, and mix by pipetting up and down. Immediately (see Note 7 ) transfer the tube to the preheated 50 °C thermocycler, and incubate for 1 h (see Note 8 ).

  5. 5.

    Mix the finished reaction thoroughly by flicking the tube, and transform a standard E. coli cloning strain like DH10B (see Note 2 ). Recover the transformed bacteria in LB medium at 37 °C for 60 min, spread on LB-spectinomycin agar plates, and incubate at 37 °C overnight.

  6. 6.

    Pick six colonies per pBIG1 construct, grow 5 ml cultures in LB-spectinomycin medium at 37 °C overnight, and isolate plasmid DNA using a miniprep kit.

  7. 7.

    Analyze clones by two separate restriction digests: digest each clone with SwaI to release individual expression cassettes from the vector backbone by mixing 1 μl 10× NEB 3.1 buffer, 1.2 μl DNA (typically ~100–500 ng/μl), 0.5 μl SwaI (10 U/μl), and 7.3 μl water. Incubate at 25 °C for 2 h. Separately, digest each clone with PmeI to release the generated polygene cassette from the vector backbone using 1 μl 10× NEB CutSmart buffer, 1.2 μl DNA, 0.2 μl PmeI (10 U/μl), and 7.6 μl water. Incubate at 25 °C for 2 h. Analyze both restriction digests on a 0.8% agarose gel (see Fig. 3a).

  8. 8.

    Sequence clones that show the expected restriction patterns (see Note 12 ) by Sanger sequencing using gene-specific primers.

Fig. 3
figure 3

Restriction analysis of pBIG1 and pBIG2 constructs. (a) Analytical digests of six clones of a 4 GEC–pBIG1 assembly (pBIG1c:APC3/APC6/APC7/APC12) were analyzed on a 0.8% agarose gel. Clones 1, 2, and 6 show the presence of all 4 GECs in the SwaI digest as well as a PGC of the expected size in the PmeI digest (correct clones indicated by arrows). (b) Analytical digests of four clones of a 2 PGC (4 GECs)–pBIG2ab assembly (lanes 1–4) and four clones of a 4 PGC (12 GECs)–pBIG2abcd assembly (lanes 5–8) coding for APC/C subcomplexes . Clones 1–4 show the presence of two PGCs derived from pBIG1a and pBIG1b constructs in the PacI digest, as well as the expected restriction pattern in the SwaI digest. Clones 5–7 show bands corresponding to four PGCs derived from pBIG1a, pBIG1b, pBIG1c, and pBIG1d constructs at the expected sizes in the PacI digest, as well as the expected restriction pattern in the SwaI digest (correct clones indicated by arrows). Clone 8 is incorrect, because it does not show a band for PGC2 in the PacI digest and is missing the corresponding bands in the SwaI digest. Note that SwaI digests of pBIG2 constructs show an additional band at 370 bp (indicated by an asterisk) that does not represent a GEC but corresponds to sequences between PGCs

Full size image

3.4 Cloning of pBIG2 Constructs Coding for Up to 25 Subunits

  1. 1.

    Choose up to five compatible pBIG1 constructs containing the GECs to be combined and a suitable pBIG2 vector (e.g., pBIG1a, pBIG1b, pBIG1c can be combined on pBIG2abc; see Fig. 2), and set up a PmeI digest to release the polygene cassettes (see Note 13 ): mix 33 ng PmeI-digested pBIG2 vector (see Subheading 3.1, step 3) with a 5× molar excess of each pBIG1 plasmid to be combined in a volume of 8 μl (see Note 14 ). Add 1 μl NEB CutSmart buffer and 1 μl PmeI and mix by pipetting. Incubate the digestion at 37 °C for 90 min.

  2. 2.

    Preheat a thermocycler block to 50 °C. Perform the Gibson assembly reaction by adding 10 μl 2× Gibson assembly master mix on ice and mix by pipetting up and down. Immediately transfer the tube to the preheated 50 °C thermocycler block and incubate for 1 h (see Notes 7 and 8 ).

  3. 3.

    Mix the finished assembly reaction thoroughly by flicking the tube, and transform a standard E. coli cloning strain such as DH10B (see Note 2 ). Recover transformed bacteria in LB medium at 37 °C for 60 min, spread on LB-chloramphenicol agar plates, and incubate at 37 °C overnight.

  4. 4.

    Pick 2–6 colonies per pBIG2 construct, grow 5 ml cultures in LB-chloramphenicol medium at 37 °C overnight, and isolate plasmid DNA using a miniprep kit.

  5. 5.

    Analyze clones by two separate restriction digests: digest each clone with SwaI to release individual expression cassettes by mixing 1 μl 10× NEB 3.1 buffer, 2.5 μl DNA (typically ~100–500 ng/μl), 1 μl SwaI (10 U/μl), and 5.5 μl water. Incubate at 25 °C for 2 h. Separately, digest each clone with PacI to release pBIG1-derived polygene cassettes by mixing 1 μl 10× NEB CutSmart buffer, 0.8 μl DNA, 0.5 μl PacI (10 U/μl), and 7.7 μl water. Incubate at 37 °C for 2 h. Analyze the SwaI digest on a 1.0% agarose gel and the PacI digest on a 0.6% agarose gel (see Fig. 3b). Select clones that show the expected restriction patterns. Note that SwaI digestion of pBIG2 constructs gives an additional band at 370 bp, corresponding to sequences between the polygene cassettes.

3.5 Generation of Recombinant Baculoviruses from biGBac Constructs

Any biGBac construct (pLIB, pBIG1, pBIG2) can be used to generate recombinant baculoviruses using Tn7 transposition. Here, a protocol is given for Tn7 transposition of cassettes from biGBac vectors onto the EmBacY baculoviral genome, which contains YFP as a fluorescent reporter [14].

  1. 1.

    Transform competent DH10-EmBacY cells with a generated biGBac construct (pLIB-, pBIG1-, or pBIG2-derived). Electroporation is recommended for constructs with a plasmid size >15–20 kb. Recover transformed bacteria in LB medium at 37 °C for 6 h. Plate the recovered bacteria on blue-white selection LB-agar plates containing X-gal, IPTG, kanamycin, tetracycline, and gentamicin. Incubate at 37 °C for 24–48 h until blue and white colonies can be clearly distinguished. Grow a white colony in LB medium containing kanamycin, tetracycline, and gentamicin at 37 °C for 16 h.

  2. 2.

    Isolate bacmid DNA by alkaline lysis using buffers from a Miniprep kit and isopropanol precipitation: resuspend the bacterial pellet in 250 μl P1, lyse with 250 μl P2, and neutralize with 350 μl N3. Clear the lysate by centrifugation at 20,000 × g for 10 min. Mix 750 μl supernatant with 750 μl isopropanol, and invert and incubate on ice for 5 min. Pellet the DNA precipitate by centrifugation at 20,000 × g, 20 min, 4 °C. Wash the DNA pellet with 70% ethanol, and centrifuge at 20,000 × g, 5 min, 4 °C.

  3. 3.

    In a laminar flow hood, remove the supernatant, air-dry the DNA pellet, and resuspend in 40 μl sterile water. Transfect Sf9 insect cells, amplify the baculovirus , and express protein using standard procedures.

4 Notes

  1. 1.

    The Gibson assembly reactions can also be performed using a homemade 1.33× Gibson assembly master mix or by mixing individual components as described [9, 10].

  2. 2.

    The Gibson assembly reaction products from the biGBac assembly steps can be transformed into any standard E. coli cloning strain. For assembly products with a size >15 kb, electroporation is recommended, because chemical transformation can show a bias for smaller incorrectly assembled by-products. For chemical transformation, use, e.g., 5 μl reaction product to transform 50 μl chemically competent cells. For electroporation, use, e.g., 0.4 μl reaction product for 40 μl electrocompetent cells.

  3. 3.

    Complete linearization of biGBac cloning vector stocks is crucial to avoid empty vector colonies.

  4. 4.

    PmeI sites (GTTTAAAC) are rare, but must be removed if present in a cDNA sequence, because they are incompatible with the second assembly step. To remove a PmeI site from a cDNA sequence, a silent mutation can be introduced by cloning the cDNA into pLIB using two PCR fragments that overlap with a Gibson overhang (~20 bp; Tm > 50 °C), which contains the silent mutation. While PmeI is the only site that needs to be removed for the biGBac assembly procedure, it can be useful to check cDNA sequences at this point also for the presence of SwaI (ATTTAAAT) and PacI (TTAATTAA) sites, because they will produce additional fragments on analytical agarose gels when analyzing multigene constructs later in the assembly procedure. There is no need to remove SwaI or PacI sites.

  5. 5.

    Small tags, e.g., His-, StrepII-, or Flag-tags, can typically be encoded on a primer. To introduce larger tags or fusion proteins, generate an additional PCR product that encodes the tag or fusion protein and overlaps with the cDNA sequence with a Gibson overhang (~20 bp; Tm > 50 °C). Separate PCR products should have a minimum size of ~100 bp.

  6. 6.

    When using gel-extracted DNA in a Gibson assembly reaction, we recommend to limit the amount of gel-extracted DNA to 5 μl in a 20 μl Gibson reaction to avoid recovering a low number of colonies. If necessary, scale down the amount of DNA accordingly without changing the vector/insert ratio.

  7. 7.

    The Gibson assembly reaction mixture should be set up on ice and transferred directly from ice to 50 °C without incubation on ice or room temperature. One of the enzymatic activities in Gibson assembly reactions, T5 exonuclease, creates single-stranded DNA ends that might form secondary structures at lower temperatures, which could reduce assembly efficiency.

  8. 8.

    Alternatively, instead of a 60-min incubation at 50 °C, a two-step incubation of 25 min at 50 °C and 10 min at 64 °C can be used. Taq ligase, one of the enzymatic activities in Gibson assembly reactions, is highly active at 64 °C.

  9. 9.

    The Cas_for and Cas_rev primers work best with an annealing temperature of 65 °C. Depending on the choice of thermocycler, it might be necessary to use a higher annealing temperature if unspecific PCR by-products are observed. It can be useful to choose a more precise temperature control mode in the settings of the PCR program to avoid short drops below the specified annealing temperature (e.g., the setting “Simulate Mastercycler gradient” for Eppendorf thermocyclers).

  10. 10.

    The high-cutoff binding buffer is used to avoid binding of potentially present primer dimers and short unspecific PCR products <300 bp to the column material. Binding to the column material also depends on the pH, which can vary depending on the used PCR buffer. Addition of 10 μl 3 M sodium acetate pH 5.0 to the PCR product before purification can improve recovery in combination with certain PCR buffers. Typical yields are 50–200 ng/μl in 30 μl elution volume. Elution efficiency might also be increased by preheating the elution buffer to 70 °C before applying it to the column.

  11. 11.

    Note that a GEC is 577 bp bigger than the inserted cDNA sequence. High DNA concentrations of the purified GECs are important for successful pBIG1 assemblies. If necessary, scale down the amount of DNA without changing the vector/GECs ratio. For the pBIG1 assembly with four GECs shown in Fig. 3a, the following components were used.

Component

Generated by

Size (bp)

c (ng/μl)

m 5× excess (ng)

V (μl)

pBIG1cSwaI

SwaI digest

5915

44

100

2.27

GEC1

CasI_for/CasI_rev

3052

226

258

1.14

GEC2

CasII_for/CasII_rev

2440

135

206

1.53

GEC3

CasIII_for/CasIII_rev

2275

132

192

1.46

GEC4

CasIV_for/Casω_rev

835

81

71

0.87

Water

    

2.73

  1. 12.

    Typically analyzing six clones of a pBIG1 assembly is sufficient to find correct clones. In some cases, it can be necessary to analyze more clones. If unspecific smaller PCR by-products seem to get incorporated into the assembly, try increasing the annealing temperature in the PCR and check the purity of the affected pLIB template. In certain cases, especially when several GECs are large and a correct clone is difficult to obtain, it can be useful to distribute the large GECs over different pBIG1 constructs, which will be combined on the pBIG2 level.

  2. 13.

    “Empty” pBIG1 vectors can also be used as placeholders in pBIG2 assemblies. For example, if expression cassettes present on pBIG1a and pBIG1c are to be combined, an “empty” pBIG1b plasmid can be used and the three plasmids combined on pBIG2abc.

  3. 14.

    Use a 5× molar excess of each pBIG1 construct over the PmeI-linearized pBIG2 vector. For example, for the pBIG2 assembly with 4 PGC (12 GECs) shown in Fig. 3b lanes 5–8, the following components were used.

Component

Plasmid size (bp)

c (ng/μl)

m 5× excess (ng)

V (μl)

pBIG2abcdPmeI

5670

36

33

0.92

pBIG1a-PGC1

13150

164

383

2.33

pBIG1b-PGC2

11900

223

346

1.55

pBIG1c-PGC3

14500

367

422

1.15

pBIG1d-PGC4

11100

241

323

1.34

Water

   

0.71