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Does anyone know the optimum length of overlap your primers should have to your vector for gibson assembly. Most manufacturers say to do at least 20 bp on both side but don't give a maximum. Sometimes I add 30-35 bp on both I don't know if that hinders efficiency.
This tutorial is an aggregation of the lessons/tips/tricks I have learned while using Gibson cloning for dozens of diverse cloning projects. It is intended to supplement available protocols with some advice and warnings that I hope can save you time with your assemblies.
- Gibson assembly allows for seamless cloning, pretty easily.
- Assemblies are independent of sequence, and you are not restricted to use of restriction enzyme cut sites.
- Assemblies are scarless.
- You can assemble multiple pieces, from multiple DNA sources (plasmids, genomes, etc.)
- It can be used for site directed mutagenesis: NEB guide
- The efficiency drops as the assembly size increases (>8 kb starts to become a problem) and as the number of pieces increases (3-4 is ok, but I haven't tried more). It is also lower when cloning toxic genes.
- Design oligos to yield 20 - 100 bp overlapping linear DNA segments
- Clean DNA fragments (column cleanup, or gel if necessary)
- Use Gibson Assembly Mix (now commercially available)
- Electroporation is usually used to provide higher yield.
For a thorough discussion on the construction of primers for use in Gibson Assembly, please see the following publication: http://www.ncbi.nlm.nih.gov/pubmed/21601685.
A note on primer design = Try to design your junctions to be in between the origin of replication or the selectable marker on the plasmid that you want to make. This will reduce your number of false-positives.
Optimum gibson assembly overlap length - Biology
The tool designs primers that are 60 nucleotides (nt) in length. Each primer includes 20 nt of gene- specific sequence for template annealing and 30-40 nt of overlap sequence, which will be used to generate homologous overlap between two adjacent fragments.
40bp homologous sequences
The Gibson Assembly® Primer Design Tool will generate primers used for adding homologous overlaps to fragments, allowing for efficient assembly. Primers are fixed at 60 nucleotides in length, and include 20 nucleotides of gene-specific sequence for template annealing. Between the vector and insert junction, you may incorporate a sequence motif, up to 10 nt long, to allow for restriction digestion. As a result, 30 to 40 bp of homologous overlap may be generated after PCR amplification. The size of overlap is sufficient to support one fragment assembly up to 10 kb, or up to 15 fragments with inserts up to 1 kb each. Extending the homologous overlap is recommended for the assembly of large genes.
DNA assembly and cloning in an overnight run with the BioXp ™ 3200 system
The BioXp ™ 3200 system is an automated personal genomic workstation that builds and clones DNA fragments in a process that is virtually hands-free. In an overnight run, the instrument generates cloned DNA from custom-designed oligonucleotide pools and reagents engineered from sequence information. Here we discuss highlights and advantages of the BioXp system and the two modules currently available, the assembly module and the assembly-and-cloning module.
As the scale of genetic analysis has trended away from single-gene studies and toward gene-family, genomic and metagenomic studies, the demand for large-scale DNA-synthesis services and technologies has grown. Moreover, with newer cloning and sequencing technologies, the pace of molecular biology research is accelerating. Synthetic Genomics, Inc., has developed the BioXp system (Fig. 1), available through SGI-DNA, as an in-house laboratory tool to address the increased interest and demand for rapid DNA synthesis. With an assembly module, the BioXp system generates high-quality, linear DNA fragments from custom-designed oligonucleotide pools and reagents across a meaningful section of the complexity continuum in an overnight run. Now, with the introduction of the assembly-and-cloning module, the BioXp system has the additional capability to deliver cloned DNA from a custom DNA sequence in an overnight run (Fig. 2).
The BioXp 3200 system, a genomic workstation.
The assembly module delivers linear DNA fragments. The assembly-and-cloning module delivers DNA cloned into a plasmid vector. Both modules build DNA in an overnight run.
Since its launch in early 2015, commercial and research laboratories have been reaping the advantages of the BioXp system, whether in individual laboratories or in core facilities. On-site access to the automated BioXp system liberates researchers from the time-consuming, tedious steps needed to obtain DNA fragments, allowing them to instead focus on new discoveries and DNA analytics.
The BioXp assembly-and-cloning module
With the assembly module, the BioXp system builds linear, blunt-end, double-stranded DNA fragments. Now, with the assembly-and-cloning module, the instrument has the added capability to build and clone DNA fragments of interest into the SGI-DNA pUCGA 1.0 vector. The pUCGA 1.0 clones generated by the BioXp system are immediately ready for transformation and further downstream analysis.
BioXp system pUCGA 1.0 clones
DNA clones obtained from the BioXp system consist of a DNA fragment of interest (400–1,800 bp, 40–60% GC content) cloned into the 2.7-kb pUCGA 1.0 vector. The pUCGA 1.0 vector map and additional information are available at http://sgidna.com. With Gibson Assembly ® technology, homologous overlap regions are automatically designed into the termini of the DNA fragments and are present in the pUCGA 1.0 vector to facilitate cloning. These homologous regions, referred to as GA ends, consist of 30 bases that have minimal sequence homology to naturally occurring genes. Highly accurate and robust automated cloning on the BioXp system is performed using the Gibson Assembly method (Fig. 3a). Clones obtained from the BioXp system are ready for transformation and subsequent downstream analysis.
(a) Overview of Gibson Assembly cloning on the BioXp 3200 system with a BioXp fragment and the pUCGA 1.0 vector. (b) High cloning efficiency from the BioXp 3200 system. As expected, the highest cloning efficiencies were achieved with the smallest DNA fragments. The total cloning efficiency (CE) for full-length inserts was calculated using the following formula: CE (%) = (Number of white colonies/Total number of colonies) × (Number of full insert colonies/Total number of colonies) × 100. The sample number (n) is shown above each respective bar error bars represent ±s.d.
Transforming pUCGA 1.0 clones
Once clones have been collected from the BioXp system deck after an assembly-and-cloning run, transformation is the next downstream step required for error-free clone identification and further analysis. The pUCGA 1.0 vector contains lacZ (the gene encoding the N-terminal fragment of β-galactosidase), which is disrupted by insertion of the BioXp DNA fragment, allowing for blue-white screening of recombinant clones. To assess clone quality, we performed transformation and calculated the cloning efficiency of pUCGA 1.0 clones. Briefly, an assembly-and-cloning run was repeated 13 times on five different BioXp instruments. Twenty clones were randomly collected from the instruments after each run, transformed into TransforMax ™ EPI300 ™ electrocompetent Escherichia coli, and plated onto LB plates containing 100 μg ml −1 carbenicillin with 40 μg ml −1 X-Gal and 0.1 mM isopropyl β-D-1-thiogalactopyranoside. After an overnight incubation, colonies were counted, picked and grown overnight, and plasmid DNA was prepared.
High cloning efficiency of pUCGA 1.0 clones
The cloning efficiency of constructs generated with the BioXp assembly-and-cloning module is shown in Figure 3b. The overall cloning efficiency of full-length pUCGA 1.0 clones was 83%. Additionally, we grouped clones according to fragment size for further cloning-efficiency analysis. As expected, the highest cloning efficiency (>90%) was observed for the smallest DNA fragments (<900 bp).
Full-length inserts were identified from double enzyme digestion with XbaI and BglII (Fig. 3a), which leaves a partial or full GA end sequence intact at the terminus of the BioXp fragment. For excision of only the fragment of interest from pUCGA 1.0, the BioXp fragment may be pre-engineered with restriction enzyme sites internal to the GA ends. Alternatively, PCR amplification with a high-fidelity DNA polymerase may be used to isolate the fragment of interest or to subclone the fragment in an alternate vector (e.g., an expression vector).
The BioXp system brings a new, rapid, automated method of DNA synthesis and cloning directly to the laboratory benchtop. The instrument can currently assemble and clone 24 DNA fragments of interest simultaneously in an overnight run. The pUCGA 1.0 DNA clones obtained from the BioXp system exhibit high cloning efficiencies—greater than 90% for DNA fragments less than 900 bp in length, and 83% overall. Laboratories using the BioXp system have the capability for virtually hands-free assembly and cloning of genes into vectors in an overnight run.
Modern cloning methods are independent from restriction enzyme recognition sites. However, nearly all current cloning methods still require the introduction of overlaps by PCR, which can introduce undesired mutations. Here, we investigated whether overlaps needed for DNA assembly can be synthesized in situ and we tested if the de novo synthesis of sequences can be simultaneously combined with the assembly of larger double-stranded DNA fragments. We showed in a set of 44 cloning experiments that overlaps of 20 bp needed for DNA assembly can be synthesized in situ from single-stranded oligonucleotides. Short sequences of 30–255 bp can be synthesized from single-stranded oligonucleotides concurrently with DNA assembly, and both techniques can be combined. The assembly of similar constructs by state-of-the-art techniques would have required multiple rounds of cloning or tedious sample preparations, whereas our approach is a one-step reaction.
Optimum gibson assembly overlap length - Biology
15 bps) than that generally recommended for SLIC/Gibson/CPEC/SLiCE (
25 bps), applying the SLIC/Gibson/CPEC/SLiCE methods to DNA fragments optimized for GeneArt® Seamless Cloning may not be successful. As described in the SLIC/Gibson/CPEC step-by-step example , it is necessary to modify the j5 design parameters to optimize assemblies for GeneArt® Seamless Cloning. For more information, see the GeneArt® Seamless Cloning documentation on the Life Technologies website.
In-Fusion® Cloning is a proprietary assembly methodology developed by Clontech . This assembly method uses the same types of DNA starting materials as those used for SLIC/Gibson/CPEC/SLiCE (described above), and results in the same final product. One key difference is that the recommended overlap length is only 15 bps (like GeneArt® Seamless Cloning, described immediately above, except it operates at 50 °C like Gibson), which may prove advantageous over SLIC/Gibson/CPEC/SLiCE from the standpoint of requiring shorter/cheaper DNA oligos and enabling combinatorial assembly designs with sequence diversity close to the ends of the sequence fragments to be assembled. On the other hand, a shorter overlap length may reduce assembly specificity, and depending on the assembly mechanism (proprietary), high self complementarity or strong single stranded DNA secondary structure in the overlap region may prove more problematic than for SLIC/Gibson/CPEC/SLiCE. Since the overlap length is shorter (
Results and Discussion
Simultaneous Assembling of One dsDNA Fragment and the De Novo Synthesis of an Additional Sequence
Our initial task was to assemble a set of constructs in which a gene of interest was fused to a target sequence at the beginning of the gene. However, the size of the fusion (3 bp) made it difficult to introduce these sequences in a primer for PCR amplification. Therefore, we tested whether the simultaneous de novo synthesis of a sequence and assembly of the gene of interest is possible with one-step isothermal assembly. 3,4 In the state of the art, these methods can be used either to assemble multiple overlapping dsDNA molecules or, alternatively, to synthesize de novo DNA molecules from overlapping oligonucleotides. The combination of both in a one-step reaction has not yet been described.
We tested this approach by cloning 47 constructs combining six different promoters, four different N-terminal fusions, and five genes (Supporting Information Figure 1). Of the 120 theoretically possible constructs, we chose 47 for cloning. The library of 47 constructs was assembled in a miniTn4001-Puro-1 backbone (GenBank accession no. <"type":"entrez-nucleotide","attrs":<"text":"KC816623","term_id":"590309357","term_text":"KC816623">> KC816623). The constructs consisted of a PCR insert (dsDNA between 500 and 2700 bp) and a de novo sequence (between 30 and 255 bp). These de novo sequences were built from combinations of two to eight oligonucleotides of around 60 bp with an overlap of 20 bp. For the complete experiment, 77 oligonucleotides were used (Supporting Information Table 2). The de novo sequence contained the promoter and a short sequence that should be fused to the PCR insert. In the first round of cloning, we obtained 29 out of 47 constructs with the correct sequence. After two more rounds of transformation and screening, we obtained 42 out of 47 constructs (Supporting Information Table 1). We reused the initial assembly reaction for the second and third rounds of transformation and colony screening. Two out of four constructs with seven oligonucleotides were not obtained with the correct sequence, even though both constructs with eight oligonucleotides were obtained with the correct sequence. We observed background in the transformation, partly because of recircularization of the vector (32% of all colonies) as well as the generation of an unknown product during the assembly process (29% of all colonies). The unknown product is a consequence of the instability of the vector backbone (minitransposon vector) and was also detected during conventional cloning using this vector. Nonetheless, this strategy enabled a convenient one-pot assembly of constructs that would be difficult to prepare in any other way. We evaluated colony PCR hits and error rates in detail for the constructs obtained in the third round of the transformation (Table 1). To increase transformation efficiencies for these constructs, the assembly reaction mixtures were purified by MinElute columns before transformation. Twelve colonies for each construct were screened, with a hit rate of 63% in the colony PCR. However, the overall hit rate was only 40.5%, partially because of the problematic backbone. For each construct, we selected four colonies that showed a hit in the colony PCR, and we sequenced the construct. On average, we obtained a correct sequence for 50% of the clones. However, the individual rates for each construct varied from 25 to 100% of correct sequences. As a general trend, longer synthesized stretches contained more errors. This is not surprising because the synthesis of oligonucleotides is error-prone and the errors accumulate with an increasing number of oligonucleotides used for a construct. The errors observed were 14 insertions, five mutations, and two deletions all were located in the part synthesized by oligonucleotides. In the screening of the complete library, only one truncation and one mutation were detected in the parts introduced as dsDNA PCR products.
construct hit rate colony PCR (%) sequencing result mutated/failed/correct correct clones (%) DNA synthesis length errors total error per bp insertions/deletions/mutations 13 58.33 3/0/1 25.00 87 5 0.057 4/1/0 15 41.67 2/0/2 50.00 114 2 0.017 2/0/0 22 83.33 1/0/3 75.00 96 2 0.020 2/0/0 19 66.67 1/1/2 50.00 69 1 0.014 1/0/0 30 66.67 2/0/2 50.00 96 3 0.031 1/0/2 31 83.33 3/0/1 25.00 96 5 0.052 3/0/2 36 41.67 0/0/4 100.00 46 0 0 0/0/0 46 75.00 1/0/3 75.00 73 1 0.013 0/0/1 41 58.33 2/1/1 25.00 52 9 0.173 1/8/0 total 63 15/2/19 52 729 21 0.03 14/2/5
In Situ Generation of Overlaps from Oligonucleotides
After observing that sequence synthesis from oligonucleotides and DNA assembly can be done simultaneously using a one-step isothermal assembly master mix, we investigated whether the overlaps needed for assembly could be added simultaneously in situ.
To synthesize the overlaps necessary for the enzymatic assembly in situ, we added four different oligonucleotides to the enzymatic assembly mix. Each oligonucleotide overlaps 20 bp with the insert (the bla gene including the promoter, PCR amplified from pJET 1.2, GenBank accession no. <"type":"entrez-nucleotide","attrs":<"text":"EF694056.1","term_id":"149999561","term_text":"EF694056.1">> EF694056.1, was used as template) and 20 bp with the linearized vector pETM14 5 (Figure (Figure1a) 1 a) (GenBank accession no. <"type":"entrez-nucleotide","attrs":<"text":"KC816624","term_id":"590309379","term_text":"KC816624">> KC816624). After transforming Top 10 E. coli competent cells with 1 μL of the assembly mix, 748 positive colonies were obtained on average.
(a) Schematic representation of the oligonucleotides used for the in situ generation of overlaps. The oligonucleotides are shown with respect to the assembled dsDNA fragments. The bar indicates the size of the oligonucleotides (they are not to scale with the overlapping regions). (b) Effect of oligonucleotide concentration on the number of colonies obtained after transformation as well as the percentage of positive colonies obtained (c) Number of colonies obtained depending on different combinations of oligonucleotides as well as the percentage of positive colonies. The percentage of positive colonies was determined by dividing the colony count on a plate with ampicillin (AMP) by the colony count on a plate with kanamycin (KAN). The oligonucleotide numbers correspond to those in panel a.
We plated transformed cells on agar plates containing either ampicillin or kanamycin as the selective agent. This allowed us to distinguish between background colonies and colonies containing our insert on a global scale without the need to use colony PCR. We observed that two oligonucleotides can be sufficient for the assembly (Figure (Figure1c), 1 c), and it appears that oligonucleotide 2 is essential for assembly, whereas oligonucleotides 3 and 4 are exchangeable. We have no explanation for this, and we observed different dependencies on other occasions (data not shown). We hypothesize that properties of the individual oligonucleotide sequence, synthesis, or preparations are the determining factor. Therefore, we recommend using as a standard procedure all four stitching oligonucleotides because this provides the most robust condition for the successful assembly of the construct. The optimal concentration of oligonucleotides in the final master mix was around 45 nM (Figure (Figure1b). 1 b). Very high concentrations of oligonucleotides ( nM) inhibited the DNA assembly step as well as reduced the efficiency of the transformation. Overall, we demonstrated that it is possible to in situ synthesize overlaps in a one-step isothermal assembly reaction, and we found robust parameters for this assembly. This facilitates the high-throughput cloning of the same fragment into many nonstandard vectors by exchanging only the stitching oligonucleotides.
We observed that isothermal assembly mixtures containing only the linearized vector yielded significant amounts of colonies, whereas the linearized vector without isothermal assembly or in the absence of Taq ligase yielded no colonies. We obtained the same results with a commercial version of the isothermal assembly mixture obtained from NEB in combination with a commercial linearized vector containing a death gene (pJET 1.2). The recircularized assembly products are truncated versions of the original vector and not a simple religation of the linearized vector. This is known to many in the field. It poses no problem in standard cloning applications because the rate of positive clones in a normal assembly is close to 90%. The background is observed only when the assembly fails or in the negative control of the vector. This is a drawback when assembly conditions have to be optimized against a negative control. Therefore, we recommend optimizing the assembly conditions using an antibiotic resistance gene as the insert or planning a colony-screening scheme with sufficient throughput.
Assembly of Two Inserts by in Situ Generation of Overlaps and De Novo Assembly of a Promoter and RBS (156 bp)
After demonstrating that the in situ synthesis of overlaps from oligonucleotides is possible and that it can be combined with the assembly of dsDNA fragments, we tested whether it is possible to combine the two methods and clone multiple fragments (Figure (Figure2). 2 ). For this, we chose to clone the importin-α (1.6 kb) and importin-β (2.6 kb) genes in the pCDF-Duet vector while building a 156 bp spacer in between them composed of a T7 promoter and modified RBS (GenBank accession no. <"type":"entrez-nucleotide","attrs":<"text":"KC816625","term_id":"590309400","term_text":"KC816625">> KC816625). To achieve this, three different oligonucleotide concentrations were tested in the final master mix: 550, 55, and 5.5 nM. As negative controls, we used a one-step isothermal assembly master mix without Taq ligase and one complete master mix missing the oligonucleotides (Supporting Information Table 4). We screened 12 colonies of the assembly mix with 55 nM oligonucleotides for the presence of both inserts, and we found eight colonies with a PCR signal for importin-α and four colonies for importin-β. Three of the colonies were positive for both inserts. The plasmid DNAs of the double-positive clones were purified, and no errors were detected after sequencing.
Schematic workflow and oligonucleotide design. (a) General workflow. dsDNA fragments and a linearized vector are obtained. Subsequently, the fragments, the vector, and the oligonucleotides are added to the one-step isothermal assembly master mix. (b) Distribution of the oligonucleotides on the construct for the assembly of importin-α (1.6 kb) and importin-β (2.6 kb) genes into the pCDF-Duet vector. A 156 bp spacer was synthesized in between them, which was composed of a T7 promoter and a modified RBS. The overlap between the individual oligonucleotides and the vector and DNA fragments is 20 bp.
We obtained 26 colonies in the assembly of the negative control without oligonucleotides and 10 in the negative control without ligase. Sequencing results showed that the background is the result of a misprimed product of the PCR linearization of the backbone. This product produced two homologous ends that were able to combine during the reaction. Such byproducts can be easily eliminated by PCR screening and do not affect the overall convenience of this method.
We have shown that the synthesis of sequences from single-stranded oligonucleotides can be combined with the assembly of DNA fragments into a vector. We also demonstrated that the overlaps needed for DNA assembly can be synthesized in situ during the assembly. By combining these two approaches, we could assemble, in a one-step reaction, two genes flanking a de novo built-up sequence.
This technique is also advantageous for individual constructs when de novo sequences have to be constructed that are too large to be introduced in a primer. For example, with our technique, a secretion signal of 30 amino acids or more can be easily synthesized in a one-step cloning reaction. By exchanging only individual oligonucleotides, mutations can be introduced in the amino acid sequence to investigate their effect on the phenotype.
The in situ synthesis of overlaps has multiple applications. First, it can be used to subclone dsDNA fragments with the versatility of one-step isothermal assembly cloning while omitting the need for any PCR reaction. Another application is in the generation of libraries for expression screening in which two genes need to be cloned in many vectors while varying the order of the genes, the promoters, and optimized RBS sites. The order of the genes can be easily changed in the in situ assembly of the overlaps by exchanging only the overlapping oligonucleotides. In the same way, a set of RBS sites can be screened or new promoters can be tested. Compared to a standard cloning, the advantages of this technique become greater with increasing sample numbers because oligonucleotides can be reused between constructs. The construction of combinatorial libraries using the isothermal assembly mixture have been described earlier. 6 However, this and other approaches depend on common linker sequences between the assembled parts, which are unnecessary in our approach. Unfortunately, we were not able to test our approach for the simultaneous assembly of a library in a single reaction mixture similar to that described by Ramon and Smith. 6
We evaluated the error rates and location of errors for a small subset of the cloned library. The error rate per base pair for the synthesized stretches is about 1 order of magnitude larger than the error rate reported for the underlying technique by Gibson et al. 4 Although we cannot directly explain this, it is worth noting that the oligonucleotides used came from a different vendor then in the original study (Sigma in our case and IDT in the case of Gibson et al. 4 ).
We have generated a library of 42 fusion genes in single-step reactions using a new approach on the basis of the one-step isothermal assembly cloning technique. This avoids a large number of PCR amplifications and multiple intermediate cloning steps. We show that sequences between 30 and 255 bp can be synthesized during the assembly of a vector with a PCR fragment of the size range between 500 and 2700 bp. The design and assembly of the constructs was very easy compared to other state-of-the-art methods and is compatible with automation. Therefore, we anticipate that this simple approach, when combined with rigorous screening methods, can be easily implemented in many applications and could be widely used in both molecular and synthetic biology.
Producing yeast expression cassettes
Traditionally, generating expression cassettes for yeast transformation can take 5 days. NEBuilder shortens that time to one day by simplifying the protocol to a single DNA assembly reaction. DNA fragments with the desired promoter, selectable marker, and target gene, for example, are amplified with overlapping regions of homology and inserted into a linearized cassette, ready for transformation into yeast that same day.
The field of synthetic biology has led to advances in biofuel generation, green chemistry and understanding the minimal genome. Many of these techniques require more complex DNA assemblies and improvements in these methodologies offer streamlined and simplified alternatives to traditional cloning. NEBuilder addresses the limitations associated with otherwise more complicated assembly approaches, and opens the door for new opportunities in the field.
Gibson Assembly® is a registered trademark of Synthetic Genomics, Inc.
One or more of these products are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc.
Many thanks to our guest blogger Lydia Morrison from New England Biolabs.
Lydia Morrison is a biochemist by training and a content marketer by profession.