Synthetic DNA Assembly Using Golden Gate Cloning and the Hierarchical Modular Cloning Pipeline

Abstract

Methods that enable the construction of recombinant DNA molecules are essential tools for biological research and biotechnology. Golden Gate cloning is used for assembly of multiple DNA fragments in a defined linear order in a recipient vector using a one‐pot assembly procedure. Golden Gate cloning is based on the use of a type IIS restriction enzyme for digestion of the DNA fragments and vector. Because restriction sites for the type IIS enzyme used for assembly must be present at the ends of the DNA fragments and vector but absent from all internal sequences, special care must be taken to prepare DNA fragments and the recipient vector with a structure suitable for assembly by Golden Gate cloning. In this article, protocols are presented for preparation of DNA fragments, modules, and vectors suitable for Golden Gate assembly cloning. Additional protocols are presented for assembly of defined parts in a transcription unit, as well as the stitching together of multiple transcription units into multigene constructs by the modular cloning (MoClo) pipeline. © 2020 The Authors. Basic Protocol 1: Performing a typical Golden Gate cloning reaction Basic Protocol 2: Accommodating a vector to Golden Gate cloning Basic Protocol 3: Accommodating an insert to Golden Gate cloning Basic Protocol 4: Generating small standardized parts compatible with hierarchical modular cloning (MoClo) using level 0 vectors Alternate Protocol: Generating large standardized parts compatible with hierarchical modular cloning (MoClo) using level –1 vectors Basic Protocol 5: Assembling transcription units and multigene constructs using level 1, M, and P MoClo vectors

Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

“…The enzyme BsaI (isoschizomer: Eco31I) with the non-palindromic recognition/hydrolysis site is active at both 37°C and 50°C, has a recognition site of 6 bp separated by one random nucleotide from the site of hydrolysis and generates a 4 bases overhang (5’ -GGTCTC(N)1-3’ / 3’-CCAGAG(N)5-5’, (2) all DNA parts of the same type were flanked by same pair of fusion sites, (3) recognition sequences of the restriction endonuclease BsaI were absent in all internal sequences of the selected 9-LOX fragments, (4) the pPICZαB vector backbone contained a LacZ resistance gene for negative selection introduced by T4 polymerase and ligase buffer without using ligase. These guiding cloning principles were also similar to those described by Marillonnet and Grützner (2020). According to Jeong et al (2012), T4 cloning was used to undertake a one-step sequence and ligation independent cloning similar to this study since ligase enzyme was not used in the cloning reaction of the Lac Z gene into the digested pPICZαB plasmid vector.…”

Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

“…The enzyme BsaI (isoschizomer: Eco31I) with the non-palindromic recognition/hydrolysis site is active at both 37°C and 50°C, has a recognition site of 6 bp separated by one random nucleotide from the site of hydrolysis and generates a 4 bases overhang (5’ -GGTCTC(N)1-3’ / 3’-CCAGAG(N)5-5’, (2) all DNA parts of the same type were flanked by same pair of fusion sites, (3) recognition sequences of the restriction endonuclease BsaI were absent in all internal sequences of the selected 9-LOX fragments, (4) the pPICZαB vector backbone contained a LacZ resistance gene for negative selection introduced by T4 polymerase and ligase buffer without using ligase. These guiding cloning principles were also similar to those described by Marillonnet and Grützner (2020). According to Jeong et al (2012), T4 cloning was used to undertake a one-step sequence and ligation independent cloning similar to this study since ligase enzyme was not used in the cloning reaction of the Lac Z gene into the digested pPICZαB plasmid vector.…”

Accelerating protein design by scaling experimental characterization

“…This process appends cloning adapters for compatibility with a range of entry vectors, and automatically splits large genes into two fragments for one-pot assembly. DNA constructs are ordered as linear gene fragments from commercial manufacturers (list price $0.07/bp) and cloned into the desired receiving vector(s) by Golden Gate Assembly (GGA) ( 35 , 36 ) followed by direct transformation into an E. coli expression strain. Target vectors contain a lethal ccdB gene ( 37 ) that is excised and replaced by the design during GGA, ensuring background suppression and thus circumventing the need for colony isolation.…”

RECKLEEN: a lambda Red/CRISPR-Cas9 based single plasmid platform for fast, efficient, markerless, and scarless genome editing in Klebsiella pneumoniae

“…For this purpose, the plasmid contains a sfgfp transcription unit as dropout element upstream of the sgRNA scaffold sequence. This sfgfp can be replaced against a specific 20-nt guide spacer sequence by Golden Gate assembly 34,35 using the type IIs restriction enzyme BsaI to generate a customized functional sgRNA gene. The initial RECKLEEN plasmid design is depicted in Supplementary Figure 1A.…”

Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

“…The enzyme BsaI (isoschizomer: Eco31I) with the non-palindromic recognition/hydrolysis site is active at both 37°C and 50°C, has a recognition site of 6 bp separated by one random nucleotide from the site of hydrolysis and generates a 4 bases overhang (5’ -GGTCTC(N)1-3’ / 3’-CCAGAG(N)5-5’, (2) all DNA parts of the same type were flanked by same pair of fusion sites, (3) recognition sequences of the restriction endonuclease BsaI were absent in all internal sequences of the selected 9-LOX fragments, (4) the pPICZαB vector backbone contained a LacZ resistance gene for negative selection introduced by T4 polymerase and ligase buffer without using ligase. These guiding cloning principles were also similar to those described by Marillonnet and Grützner (2020). According to Jeong et al (2012), T4 cloning was used to undertake a one-step sequence and ligation independent cloning similar to this study since ligase enzyme was not used in the cloning reaction of the Lac Z gene into the digested pPICZαB plasmid vector.…”

Accelerating protein design by scaling experimental characterization

“…This process appends cloning adapters for compatibility with a range of entry vectors, and automatically splits large genes into two fragments for one-pot assembly. DNA constructs are ordered as linear gene fragments from commercial manufacturers (list price $0.07/bp) and cloned into the desired receiving vector(s) by Golden Gate Assembly (GGA) ( 35 , 36 ) followed by direct transformation into an E. coli expression strain. Target vectors contain a lethal ccdB gene ( 37 ) that is excised and replaced by the design during GGA, ensuring background suppression and thus circumventing the need for colony isolation.…”

RECKLEEN: a lambda Red/CRISPR-Cas9 based single plasmid platform for fast, efficient, markerless, and scarless genome editing in Klebsiella pneumoniae

“…For this purpose, the plasmid contains a sfgfp transcription unit as dropout element upstream of the sgRNA scaffold sequence. This sfgfp can be replaced against a specific 20-nt guide spacer sequence by Golden Gate assembly 34,35 using the type IIs restriction enzyme BsaI to generate a customized functional sgRNA gene. The initial RECKLEEN plasmid design is depicted in Supplementary Figure 1A.…”

Molecular cloning and recombinant protein expression of 9-Lipoxygenase gene from Solanum tuberosum in Pichia pastoris yeast cells

“…The enzyme BsaI (isoschizomer: Eco31I) with the non-palindromic recognition/hydrolysis site is active at both 37°C and 50°C, has a recognition site of 6 bp separated by one random nucleotide from the site of hydrolysis and generates a 4 bases overhang (5’ -GGTCTC(N)1-3’ / 3’-CCAGAG(N)5-5’, (2) all DNA parts of the same type were flanked by same pair of fusion sites, (3) recognition sequences of the restriction endonuclease BsaI were absent in all internal sequences of the selected 9-LOX fragments, (4) the pPICZαB vector backbone contained a LacZ resistance gene for negative selection introduced by T4 polymerase and ligase buffer without using ligase. These guiding cloning principles were also similar to those described by Marillonnet and Grützner (2020). According to Jeong et al (2012), T4 cloning was used to undertake a one-step sequence and ligation independent cloning similar to this study since ligase enzyme was not used in the cloning reaction of the Lac Z gene into the digested pPICZαB plasmid vector.…”

Accelerating protein design by scaling experimental characterization

“…This process appends cloning adapters for compatibility with a range of entry vectors, and automatically splits large genes into two fragments for one-pot assembly. DNA constructs are ordered as linear gene fragments from commercial manufacturers (list price $0.07/bp) and cloned into the desired receiving vector(s) by Golden Gate Assembly (GGA) ( 35 , 36 ) followed by direct transformation into an E. coli expression strain. Target vectors contain a lethal ccdB gene ( 37 ) that is excised and replaced by the design during GGA, ensuring background suppression and thus circumventing the need for colony isolation.…”

RECKLEEN: a lambda Red/CRISPR-Cas9 based single plasmid platform for fast, efficient, markerless, and scarless genome editing in Klebsiella pneumoniae

“…For this purpose, the plasmid contains a sfgfp transcription unit as dropout element upstream of the sgRNA scaffold sequence. This sfgfp can be replaced against a specific 20-nt guide spacer sequence by Golden Gate assembly 34,35 using the type IIs restriction enzyme BsaI to generate a customized functional sgRNA gene. The initial RECKLEEN plasmid design is depicted in Supplementary Figure 1A.…”