Background

DNA cloning tools have been improved to optimize the efficiency of cloning systems. New cloning methods were designed, such as (i) Gibson assembly, which integrates a number of DNA fragments in a single reaction using a mix of the three enzymes exonuclease, DNA polymerase, and DNA ligase (Gibson et al., 2009), (ii) Gateway, which requires specific recombination sites (atts) in both the DNA fragment and plasmids used in the two reactions steps: BP reaction—in which an entry vector, harboring a DNA fragment flanked by two attL sites, is created; and LR reaction—wherein an expression vector containing a DNA fragment flanked by attB sites is generated (Karimi et al., 2007; Invitrogen, USA); (iii) Golden Gate cloning, a method based on the use of type IIS endonucleases that have cut sites distal from their recognition sites producing overhangs; the complementary overhangs are traditionally ligated by T4 DNA ligase (Engler et al., 2008); (iv) USER cloning (Uracil-Specific Excision Reagent), which employs primers containing uracil to perform PCR reactions; the PCR product is incubated with a commercial deoxyuridine-excision enzyme to eliminate the uracil and generate 3' overhangs that allow the direct cloning of PCR fragments into a USER-compatible vector (Geu-Flores et al., 2007; Hansen et al., 2014); and (v) In-Fusion, in which four or more DNA fragments containing an overlapping of 15 bp at both ends are combined by a reaction equivalent to recombination; the ends overlap is achieved by including the 15 bp in the primers used for DNA amplification (Zhu et al., 2007).

Gateway and Golden Gate are the most used cloning systems. Both have high efficiency and accuracy. However, the drawback of Gateway is the dependency on specific commercial enzymes used for the two clonase reactions, BP and LR, which makes this technique more expensive. On the other hand, Golden Gate requires specific restriction sites in the vector as well as in the DNA fragments, which could be a limiting factor when designing constructs for a large number of genes, as the components of this method are generally not reusable (Marillonnet and Grützner, 2020). Thus, modular assembly systems have been developed to standardize the parts, or bricks (DNA fragments of interest), which is crucial for cloning construction. These modular systems facilitate cloning assembly, making it simpler, more versatile, and autonomous (Patron et al., 2015).

Here, we provide a new cloning method, Brick into the Gateway (BiG), that uses the GoldenBraid/Gate methods to assemble the bricks and the LR reaction from the Gateway system to transfer DNA fragments into any destination vector. Using the GoldenBraid system, we generated standard bricks of the attL sites into an acceptor plasmid pUPD2, which can be combined with other bricks harboring any DNA fragments through a Golden Gate reaction. After this reaction, an entry vector containing the DNA of interest flanked by attL sites is generated, allowing its transfer to a destination vector using Gateway LR clonase. BiG is a low-cost method with a short processing time and directional cloning, whose efficiency was successfully demonstrated (Ferigolo et al., 2022). Most importantly, BiG allows the use of assembled bricks in different studies due to its common syntax (Ferigolo et al., 2022).

Materials and Reagents

1. Enzymes

   1. High-fidelity DNA polymerase with proper buffer (e.g., platinum SuperFi II polymerase, Invitrogen^TM, catalog number: 12361010)

   2. Restriction enzyme cloning BsmBI-v2 with proper buffer (New England Biolabs Inc., catalog number: R0580)

   3. Restriction enzyme cloning BsaI-HFv2 with proper buffer (New England Biolabs Inc., catalog number: R3733)

   4. T4 DNA ligase with proper buffer (e.g., Thermo Fisher Scientific^TM, catalog number: EL0014)

   5. Gateway LR clonase II enzyme mix with proper buffer (Invitrogen^TM, catalog number: 11791100)

   6. GoTaq green master mix (Promega^TM, catalog number: M7122)

      
      

2. Cloning vectors

   1. Vector harboring attL sites (e.g., pENTR^TM/D-TOPO^TM)

   2. LM22_attL1 (Addgene, catalog number: 184008)

   3. LM22_attL2 (Addgene, catalog number: 184010)

   4. Universal GoldenBraid acceptor (e.g., pUPD2)

   5. Golden Gate acceptor vector (e.g., pICSL86900OD)

   6. Gateway expression vector of choice

      
      

3. Kits

   1. Gel extraction kit (e.g., QIAEX II, QIAGEN^TM, catalog number: 20021)

   2. Plasmid Miniprep kit (e.g., GeneJET, Thermo Fisher Scientific^TM, catalog number: K0702)

      
      

4. Others

   1. dNTPs, 10 mM (Thermo Fisher Scientific^TM, catalog number: R0192)

   2. PCR primers

   3. DNase- and RNase-free water

   4. PCR tubes, 0.2 mL (Axygen^TM, catalog number: 14-222-262)

   5. 6×loading dye (New England Biolabs Inc., catalog number: B7024S)

   6. 1 Kb plus DNA ladder (e.g., Invitrogen^TM, catalog number: 10787018)

   7. Agarose 1.5% gel (BioBasic, catalog number: AB0014)

   8. SYBR^TM Safe DNA gel stain (Invitrogen^TM, catalog number: S33102)

   9. Escherichia coli DH10α chemically competent cells (Thermo Fisher Scientific, catalog number: 18265017)

   10. LB plates with the appropriate antibiotic, IPTG (Fermentas, catalog number: R0392), and X-gal substrate (Fermentas, catalog number: R0401)

   11. Chloramphenicol (Sigma-Aldrich, catalog number: C0378)

   12. Esp31 (Thermo Fisher Scientific^TM, catalog number: ER0451)

   13. Eco31I (Thermo Fisher Scientific^TM, catalog number: ER0291)

   14. GoldenBraid (GB) reaction (TV = 10 µL) (see Recipes)

   15. LB medium (see Recipes)

   16. Golden Gate reactions (TV = 10 µL) (see Recipes)

   17. LR reaction (TV = 5 µL) (see Recipes)

Equipment

1. PCR cycler (Mastercycler^® nexus, catalog number: 6330000021)

2. Microwave oven

3. Gel electrophoresis chamber

4. Blue-Light transilluminator (Safe Imager^TM 2.0, catalog number: G6600)

5. NanoDrop^TM OneC Microvolume UV-Vis spectrophotometer (Thermo Fisher Scientific^TM, catalog number: 13-400-519)

6. Heat block at 42 °C

7. ColiRollers^TM plating beads, Novagen^® (Sigma-Aldrich^TM, catalog number: 71013-3)

8. Incubators at 37 °C

Software

1. Benchling R&D Cloud (https://www.benchling.com/)

Procedure