A scientific update
Issue I • 2020
Can looking to the future help preserve a historical fishery against climate change?
Keeping pace with the changing ocean to protect cod
Eva Nogales speaks about her pioneering work in cryogenic electron microscopy
Donald G. Comb Honorary Lecture Series
Pushing the limits of Golden Gate Assembly
Meet our new 100% recyclable ClimaCell® shipper
Find out how our new cell-free protein synthesis system delivers high performance and versatility
Breeding a better tomato with the NEBNext Direct® Genotyping Solution
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The global population is anticipated to eclipse 8 billion by 2025, and approach 10 billion people by 2050 (1). Along with this rate of growth comes global challenges for feeding this population, pressing the need for more efficient farming practices. This efficiency is perturbed by reduced land availability for farming, emergence of novel pathogens, diminishing table water, and observed changes in climate, generating the need for sustainable crops with improved resilience to these stresses.
Traditional crop breeding approaches rely on interbreeding, or “crossing” of plant varieties for allelic transfer to generate genetic diversity within new crop varieties. This is followed by phenotypic assessment, and subsequent backcrossing with the parental lines for selection of desired traits. These traits can be quantitative and practical, such as pathogen resistance, drought tolerance and improve-ments in crop yield, or they can be more subjective and aesthetic traits including flavor and color. While these methods are effective, they rely on plant growth, exposure to stress, and observation of the desired phenotype in order to assess the presence of the desired trait; therefore, the breeding process is greatly lengthened.
Quantitative trait locus, or QTL mapping, generates linkage information between a desired phenotype and the associated genotypic information. There are several approaches available to perform QTL mapping, but the goal is to identify a set of genetic markers that can be used in place of phenotypic information to assess whether plants are harboring the specific markers that are positive indicators for the presence of traits being selected for. The development of crop-specific databases to guide breeding programs has created a need for novel methods for genotyping plants that result from genetic crossing or backcrossing in order to guide future breeding efforts. To generate necessary genetic diversity, thousands to tens of thousands of plants are used for breeding efforts, so ideally these approaches are fast and scalable to address the throughput demand.
By Andrew Barry, MS, New England Biolabs
Traditional genotyping approaches include endpoint PCR assays, whereby only a limited number of markers for any given plant can be assayed, and input samples must be split across multiple PCR reactions, requiring high amounts of PCR consumables and specialized equipment for high-throughput sample processing. Another common option for genotyping is the use of SNP-based microarrays, which can assay hundreds of thousands of markers in parallel, yet the challenge lies in scaling the experiment for high sample numbers, because a single DNA sample per chip is required. In parallel, the creation of full genome reference sequences for many crops has increased our knowledge, and therefore, additional marker types including genomic insertions and deletions, and combinations of markers, or haplotypes, are increasingly used as phenotypic indicators. Microarrays and endpoint PCR assays are limited in their ability to solely provide information on the presence or absence of a known marker and cannot be used for discovery of novel genomic information.
The advent of next-generation sequencing has provided scientists in many research areas with a tool to understand genomic information in a cost-effective manner. The continual decreases in the cost of sequencing have made this an attractive readout that provides not only genotype information, but con-textual information of the areas surrounding target genomic loci, that can increase the types of available genomic markers, and also lead to the discovery of new markers. The efficiency of next-generation sequencing has shifted the throughput challenge further upstream, necessitating improved methods for preparing samples for sequencing analysis, in the most efficient way possible.
The development of crop-specific databases to guide breeding programs has created a need for novel methods for genotyping plants that result from genetic crossing or backcrossing in order to guide future breeding efforts.
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INTERACTIVE FIGURE 1A:
The NEBNext Direct Genotyping Solution Workflow
Final library and sequencing details
Samples are tagged with a barcode prior to pooling. Individual molecules are marked with a Unique Molecular Identifier (UMI)
Biotin baits target both strands (shown for 1 strand)
Enzymatic removal of off-target sequence
Individual samples are identified by the inline sample index and a pool index
The NEBNext Direct Genotyping Solution was developed to address the specific needs of high-throughput, targeted genotyping required for breeding applications. Developed in collaboration with industrial crop breeders, the method employs high levels of sample multiplexing along with highly efficient, capture-based enrichment of hundreds to thousands of genomic loci, to enable next-generation sequencing to be fully leveraged as a means for high-throughput, cost-effective genotyping.
In order to demonstrate the efficacy of the approach, a panel of 2309 genomic markers was developed to target SNPs from the Solanaceae Coordinated Agricultural Project (SolCAP) database (2). This panel was assayed against extracted DNA from 96 samples of the tomato crop, Solanum lycopersicum, and processed using the NEBNext Genotyping Solution before Illumina® sequencing.
Key features aiding the efficiency of the NEBNext Direct Genotyping Solution include the consol-idation of DNA fragmentation with end repair, 5´ phosphorylation, and dA-tailing into a single enzymatic step. This is immediately followed by ligation of the Sample Index, which contains a 5´ adaptor to barcode the samples. These two steps represent the only workflow steps where samples are processed individually, as sample pooling immediately follows. By pooling samples (up to 96) prior to capture-based enrichment, the processing steps are significantly reduced, and there is a vast reduction in laboratory consumables required. The 5´ adaptor also contains a 12 base pair, random sequence known as a Unique Molecular Identifier, or UMI. The UMI is used to individually index each library molecule and is used in data analysis to identify duplicate molecules that are generated during the downstream amplification processes, as well as aiding more accurate genotype calls.
The UMI is used to individually index each library molecule and is used in data analysis to identify duplicate molecules that are generated during the downstream amplification processes, as well as aiding more accurate genotype calls.
The bait hybridization step hybridizes both DNA strands using synthetic, biotinylated oligonucleotides designed against all 2,309 genomic regions harboring the loci of interest for all 96 samples, capturing a total of over 220,000 data points in a single enrichment reaction. These captured molecules are subsequently fully converted into a next-generation sequencing library, during which specificity enhancing enzymatic treatments are performed, and a second, pool-specific barcode is added to the 3´ end of molecules. This dual-indexing strategy enables further pooling prior to sequencing, maximizing the output of Illumina® sequencing. In order to assess performance across 96 samples, sequencing data was processed and aligned to the reference genome sequence, and key metrics were obtained. Analysis of the percent selected across each of the 96 samples demonstrates the method’s ability to efficiently enrich molecules containing the target SNP markers, with consistent values across samples showing > 95% of sequencing data mapping to the defined targets (Figure 2).
Final library and sequencing details
The percent of passing filter reads mapping to targeted regions demonstrates high specificity across 96 multiplexed samples using the NEBNext Direct Genotyping Solution. 25 ng of purified tomato DNA was used as input for each sample. Samples were index-tagged and pooled prior to hybridization and libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.
Mean SNP coverage across 96 pooled samples
Mean SNP coverage of 2,309 SolCAP markers across 96 samples. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and Libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.
Sequencing coverage depth observed across 2,309 marker loci within a single sample
Histogram of coverage across each of the 2,309 SolCAP markers demonstrates evenness of enrichment across targets and coverage levels sufficient for genotyping calls. These data represent enrichment of a single tomato sample pooled with 95 others prior to hybridization. 25 ng of purified tomato DNA was used for each sample. Samples were index-tagged and pooled prior to hybridization and libraries were sequenced on an Illumina MiSeq with 20 cycles of Read 1 to sequence the 12 base UMI and 8 base sample index, and 75 cycles of Read 2 to sequence the targets.
Further analysis into the ability to confidently determine the genotype of the 2,309 SNPs, demonstrates the ability of the method to produce consistent sequencing coverage at depths sufficient to assess the presence or absence of SNP markers across the 96 pooled samples (Figure 3).
A closer examination of the specific performance across targets included in the panel within a single sample shows coverage across the highest and lowest performing targeted regions within.
These data suggest that by using the NEBNext Direct Genotyping Solution, high-throughput, massively parallel enrichment of genomic loci can be achieved in an efficient manner upfront of next generation sequencing. The combined efficiency of a novel sample-preparation strategy and continued advances in next generation sequencing present a tractable solution for genotyping hundreds to thousands of genomic loci that can be employed to accelerate plant breeding programs aimed at production of new crop variants that can overcome the challenges our global population growth will continue to present.
In the NEB Expressions feature article “Over 40 years in protein expression and purification – a historical perspective” (Issue III, 2019) Har Gobind Khorana was accidentally omitted from the timeline “Advances in DNA Understanding were Foundational for Protein Overexpression” (Figure 1, page 2). Khorana published seminal work on cracking the genetic code. In 1968, he shared the Nobel Prize with Marshall Nirenberg and Robert Holley for their interpretation of the genetic code and its function in protein synthesis. This figure has been corrected in both web and PDF versions of the article available on www.neb.com. We apologize for this oversight.
1. United Nations, Department of Economic and Social Affairs, Population Division (2019). World Population 2019: Wall Chart (ST/ESA/ SER.A/434).
2. Solanaceae Coordinated Agricultural Project (SolCAP). 2011. Tomato intervarietal TA496 vs. Heinz1706 SNPs. Michigan State University, Dept. of Plant and Soil Science, East Lansing, MI. http://solcap.msu.
5. Li H. (2013) Aligning sequence reads, clone sequences and assem-bly contigs with BWA-MEM. arXiv:1303.3997v2 [q-bio.GN]6. Fulcrum Genomics, https://github.com/fulcrumgenomics/fgbio
NEBNext Direct® Genotyping Solution
The NEBNext Direct Genotyping Solution combines highly multiplexed, capture-based enrichment with maximum efficiency next generation sequencing to deliver cost-effective, high throughput genotyping for a wide variety of applications. Applicable for ranges spanning 100-5,000 markers, pre-capture multiplexing of up to 96 samples combined with dual indexed sequencing allows over 3.8 million genotypes in a single Illumina sequencing run.
NGS-based targeted genotyping for a wide range of applications
Marker Assisted Selection / Breeding Quantitative Trait Locus (QTL) Screening
Mouse Genotyping Livestock Breeding
NGS Sample Tracking
High throughput targeted genotyping for Illumina® sequencing
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Optimized Bait Design
Sample Indexing and Multiplexing
The NEBNext Direct Genotyping Solution employs a purpose-built bait designer that has been optimized to provide both highly specific capture of target loci and maximized sequencer efficiency. By designing baits independently to each target DNA strand with proximity to the target loci, shorter sequence reads can be utilized for genotyping calls. Further, by removing upstream off-target sequence, individual baits can be unambiguously linked to their corresponding sequencing read, presenting opportunities for bait optimization on a per target level and resulting in extremely uniform coverage levels across markers.
With 96 pre-capture sample indexes and 8 post-capture pool indexes available, up to 96 samples can be combined for a single capture, and 768 samples can be pooled into a single Illumina sequencing run. Additionally, a 12 bp Unique Molecular Identifier (UMI) is added prior to sample pooling and enrichment, allowing for accurate assessment of input coverage and improving the accuracy of genotyping calls. Finally, sequencing cycle numbers are optimized to sequence only the necessary target region, indexes and UMI required for marker genotyping. The NEBNext Direct Genotyping Solution is compatible with the full range of Illumina sequencers.
Marker coverage across DNA strands
Two examples of the coverage of targeted markers within a single sample from the 96-plex enrichment as visualized in the Integrative Genome Browser (IGV)1,2. Reads shown are de-duplicated using UMIs. Baits target both strands of the input DNA, as indicated by the red and blue aligning reads.
Push the Limits of Golden Gate Assembly
20+ fragment assembly now achievable with high efficiency and accuracy
TOOLS & RESOURCES
Overview of Golden Gate Assembly
< Back to Golden Gate main
The efficient and seamless assembly of DNA fragments, commonly referred to as Golden Gate Assembly (1,2), has its origins in 1996, when for the first time it was shown that multiple inserts could be assembled into a vector backbone using only the sequential (3) or simultaneous (4) activities of a single Type IIS restriction enzyme and T4 DNA Ligase. Golden Gate Assembly and its derivative methods exploit the ability of Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition sequence. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly.
With constant advances in both the development of new enzymes and research on maximizing enzyme functionality (e.g., ligase fidelity), NEB has become the industry leader in pushing the limits of Golden Gate Assembly and related methods, such as MoClo, GoldenBraid, Mobius Assembly and Loop Assembly. We have all the products and information you need to perform complex assemblies (20+ fragments) with high efficiencies, >90% accuracy and low backgrounds.
Golden Gate Assembly Workflow
In its simplest form, Golden Gate Assembly requires a Type IIS recognition site, in this case, BsaI-HFv2 (GGTCTC), added to both ends of a dsDNA fragment. After digestion, these sites are left behind, with each fragment bearing the designed 4-base overhangs that direct the assembly.
Golden Gate Assembly workflow for both simple and complex assemblies
Clone seamlessly, with no scars remaining after assembly
Perform single insert cloning in just 5 minutes using our fast protocols
Generate libraries with high efficiencies
Assemble multiple fragments (2-20+) in order, in a single reaction
Experience high efficiency, even with regions of high GC content and areas of repeats
Use with a broad range of fragment sizes (<100 bp to >15 kb)
Can be used in complex (20+) fragment assemblies
Featured Products for Golden Gate Assembly
Tools & Resources for Golden Gate Assembly
Advances in Ligase Fidelity
Expanded “assembly standards” for MoClo, GoldenBraid 2.0 and other modular Golden Gate Assembly methods
Tools to help with designing your reactions
Expanded “assembly standards” for MoClo, GoldenBraid 2.0 and other modular Golden Gate Assembly methods
For help with designing primers
Try our ligase fidelity tools for the design of high-fidelity Golden Gate assemblies
NEB Golden Gate Assembly Tool
Ligase Fidelity Viewer™ (v2)
Visualize overhang ligation preferences
Predict high-fidelity junction sets
Split DNA sequence for scarless high-fidelity assembly
Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly
Research at NEB has led to an increased understanding of ligase fidelity, including the development of a comprehensive method for profiling end-joining ligation fidelity in order to predict which overhangs have improved fidelity (1). This research has enabled complex fragment assemblies with high efficiency and >90% accuracy.
Fidelity and Bias in End-Joining Ligation: Enabling complex, multi-fragment Golden Gate DNA Assembly
1. Potapov, V. et. al. (2018) ACS Synth. Biol. 7,1, 2665-2674. PMID: 30335370
Eleven Tips For Optimizing Your Golden Gate Assembly Reactions
Check your sequences
Always check your assembly sequences for internal sites before choosing which Type IIS restriction endonuclease to use for your assembly. While single insert Golden Gate assembly has such high efficiencies of assembly that the desired product is obtainable regardless of the presence of an internal site, this is not true for assemblies with multiple inserts. Options include choosing a different Type IIS restriction enzyme to direct your assembly, or eliminating internal sites through mutagenesis. The Q5® Site-Directed Mutagenesis Kit (NEB #E0554S) and the NEB web tool NEBaseChanger work well for this purpose. Alternately, a junction point can be created at the internal site’s recognition sequence.
Orient your primers
When designing PCR primers to introduce Type IIS restriction enzyme sites, either for amplicon insert assembly or as an intermediate for pre-cloning the insert, remember that the recognition sites should always face inwards towards your DNA to be assembled. Consult the Golden Gate Assembly Kit manuals for further information regarding the placement and orientation of the sites.
Choose the right plasmid
Consider switching to the pGGAselect Destination Plasmid for your Golden Gate assembly. This versatile new destination construct is included in all Golden Gate Assembly kits and can be used for Bsa-HFv2, BsmBI-v2 or BbsI directed assemblies. It also features T7 and SP6 promoter sequences flanking the assembly site, and has no internal BsaI, BsmBI or BbsI sites. The pGGAselect plasmid can also be transformed into any E. coli strain compatible with pUC19 for producing your own plasmid preparation if so desired.
Choose the right buffer
T4 DNA Ligase Buffer works well for Golden Gate Assembly with both BsaI-HFv2 and BsmBI-v2. However, alternate buffers would be NEBuffer 1.1 for Bsa-HFv2 and NEBuffer 2.1 for BsmBI-v2, as long as supplemented with 1 mM ATP and 5-10 mM DTT.
Increase your complex assembly efficiency by increasing the Golden Gate cycling levels
T4 DNA Ligase, BsaI-HFv2 and BsmBI-v2 are very stable and survive extended cycling protocols; an easy way to increase assembly efficiencies without sacrificing fidelity is to increase the total cycles from 30 to 45-65, even when using long (5-minute) segments for the temperature steps.
Make sure your plasmid prep is RNA-free
For pre-cloned inserts/modules, make sure your plasmid prep is free of RNA to avoid an over-estimation of your plasmid concentrations.
Avoid primer dimers
For amplicon inserts/modules, make sure your PCR amplicon is a specific product and contains no primer dimers. Primer dimers, with Type IIS restriction endonuclease sites (introduced in the primers used for the PCR reactions), would be active in the assembly reaction and result in mis-assemblies.
Avoid PCR-induced errors
Do not over-cycle and use a proofreading high fidelity DNA polymerase, such as Q5® DNA High-Fidelity Polymerase.
Decrease insert amount for complex assemblies
For complex assemblies involving >10 fragments, pre-cloned insert/modules levels can be decreased from 75 to 50 ng each without significantly decreasing the efficiency of assembly.
Carefully design EVERY insert’s overhang
An assembly is only as good as its weakest junction. Research at NEB has led to an increased understanding of ligase fidelity, including the development of a comprehensive method for profiling end-joining ligation fidelity in order to predict which overhangs will result in improved accuracy. This ligase fidelity information can be used in conjunction with the NEB Golden Gate Assembly Kits (BsaI-HFv2 or BsmBI-v2) to achieve high efficiency and accurate complex assemblies. Please use the free NEB Golden Gate Assembly Tool to design primers for your Golden Gate Assembly reactions, predict overhang fidelity or find optimal Golden Gate junctions for long sequences. When working with complex assemblies (>20 fragments), refer to the ligase fidelity tools on the NEBeta Tools site.
Check for a sequence error if your assembly becomes non-functional
Be aware that occasionally a pre-cloned insert/module can become corrupted by an error during propagation in E. coli, usually a frameshift due to slippage in a run of a single base (e.g., AAAA) by the E. coli DNA Polymerase. This should be suspected if previously functional assembly components suddenly become nonfunctional.
Looking to assemble multiple DNA fragments in a single reaction? Here are some tips to keep in mind when planning your Golden Gate Assembly experiment.
View full usage guide
What is Modular Cloning?
Modular Cloning, better known as MoClo, is a Type IIS assembly method commonly used by synthetic biologists, including those from the plant and AgBio community, for the assembly of multiple biological parts to engineer new biological systems. MoClo methods provide efficient and seamless assembly and allow users to generate and reuse “parts for assemblies” so that laboratories can share assembly-ready sequences. MoClo methods depend on Type IIS restriction enzymes (such as BsaI-HFv2, BbsI/BbsI-HF and BsmBI-v2/Esp3I) that leave 4-base overhangs. NEB has extensively studied the fidelity of ligation for all possible 4-base overhangs. With this information, we are able to estimate the fidelity of any set of junctions (fusion sites).
Like Golden Gate Assembly, MoClo (and GoldenBraid 2.0) uses 3 levels of successive assembly. The community has agreed upon a set of common standard overhangs for each level as indicated below, along with predicted fidelity:
MoClo Standardized Assembly Overhangs
• Level 0 (Basic parts): ACAT, TTGT (94% fidelity)
• Level 1 (Transcriptional units): GGAG, TACT, CCAT, AATG, AGGT, TTCG, GCTT, GGTA, CGCT (93% fidelity)
• Level 2 (Multigene constructs): TGCC, GCAA, ACTA, TTAC, CAGA, TGTG, GAGC, GGGA (95% fidelity)
Expanded MoClo Standardized Assembly Overhangs
Utilizing the gathered ligase fidelity information, NEB has expanded each level of assembly overhangs without sacrificing fidelity. The expanded sets are described below:
• Level 0 (Basic parts):
ACAT, TTGT, ACTG, GCTA, CCCA, AATA, ATTC, GTGA, CGCC, AAGA, AAAC, AACG, CTGC. GACC, AAGA, AAAC, AACG, CTGC, GACC, CTAA, ACCC, TACA, GGAA, CAAG, AGAG (93% fidelity)
• Level 1 (Transcriptional units): GGAG, TACT, CCAT, AATG, AGGT, TTCG, GCTT, GGTA, CGCT, GAAA, TCAA, ATAA, GCGA, CGGC, GTCA, AACA, AAAT, GCAC, CTTA, TCCA (92% fidelity)
• Level 2 (Multigene constructs): TGCC, GCAA, ACTA, TTAC, CAGA, TGTG, GAGC, GGGA, CGTA, CTTC, ATCC, ATAG, CCAG, AATC, ACCG, AAAA, AGAC, AGGG, TGAA, ATGA (95% fidelity)
NEB Golden Gate Assembly Kit (BsmBI-v2)
The absence of internal sites in a sequence determines the choice of which Type IIS restriction enzyme to drive the assembly. For your convenience, NEB now offers two kits for Golden Gate Assembly featuring BsaI-HF-v2 or BsmBI-v2. Both kits incorporate digestion followed by ligation with T4 DNA Ligase into a single reaction, and can be used to assemble 2-20+ fragments in a single step.
NEB also offers more Type IIS restriction enzymes that any other supplier, many of which are used in Golden Gate assembly. These enzymes recognize asymmetric DNA sequences and cleave outside of their recognition sequence. BsmBI-v2 has been optimized for Golden Gate Assembly and replaces BsmBI.
NEBExpress® Cell-free E. coli Protein Synthesis System
A High Performance E. coli Cell Lysate-Based System for in vitro Protein Synthesis
Paula Magnelli, Ph.D., Haruichi Asahara,Ph.D., Julie Beaulieu, M.S., Jim Samuelson, Ph.D., Stephen Shi, Ph.D., New England Biolabs, Inc.
Cell-free protein synthesis (CFPS) systems based on bacterial cell lysates have been widely used for an array of applications. These systems offer a number of advantages; for example, the tight coupling of translation and transcription from prokaryotic cells is preserved in the lysate, rendering the protein synthesis process exceptionally efficient. Additionally, genetic manipulation can be performed on the strain in which the lysates are made to enhance its ability to generate high yields of protein. Finally, the lysates can be manufactured at a larger scale, compared to reconstituted systems. Lysate-based protein synthesis systems are conceptually simple and relatively less expensive, and have therefore been home-brewed in many laboratories over the past decades, as well as supplied by several commercial sources. These systems, however, exhibit varying levels of performance, ease of use, and often do no produce consistent results across broad size ranges and types of proteins.
The NEBExpress Cell-free E. coli Protein Synthesis System was developed using several strategies to enhance performance, ease of use, and ensure robustness. These include the use of an E. coli strain genetically engineered to maximize the stability of template DNA and RNA and the protein products, a highly optimized reaction buffer, and a stringent biomanufacturing process.
The NEBExpress Cell-free E. coli Protein Synthesis system was developed using several strategies to enhance performance, ease of use, and ensure robustness.
In this technical note, we examine how the NEBExpress Cell-free E. coli Protein Synthesis System performs in several frequently encountered applications, and demonstrate this system’s high performance and versatility.
Cell-free Protein Synthesis (CFPS) reaction
The following reagents were combined in 1.5 ml microcentrifuge tubes:
• 25 µl Protein Synthesis Buffer (2X)
• 1 µl T7 RNA Polymerase
• 1 µl RNase Inhibitor
• 12 µl NEBExpress S30 Synthesis Extract
• 2 µl DNA template (125 ng/µl)
• Water to 50 µl
Reactions were incubated with shaking for 3 hours at 37°C (unless otherwise indicated).
Green fluorescent protein (vGFP) assay
The following reagents were combined in a black 96-well plate (Corning #3165):
• 25 µl Protein Synthesis Buffer (2X)
• 1 µl T7 RNA Polymerase
• 1 µl RNase Inhibitor
• 12 µl NEBExpress S30 Synthesis Extract
• 2 µl vGFP template (125 ng/µl)
• Water to 50 µl
Plates were covered with a breathable seal, and reactions were incubated with intermittent shaking for 5 hours at 37°C (unless otherwise indicated). Fluorescence detection: emission at 513 nm, excitation at 532 nm, 6 flashes/read.
CFPS samples were diluted 1:10 in water. 5 µl of solution was combined with 200 µl of 5 mM 2-nitrophenyl β-D-galactopyranoside (Sigma)
in 50 mM sodium phosphate buffer pH7.3. Samples were incubated at 37°C for 30 min. Free 2-nitrophenol was measured by absorption
at 420 nm.
CFPS samples (1-10 µl) were mixed with 200 µl of 40 µM 4-Methylumbelliferyl-β-D-N,N’,N’’-triacetylchitotrioside (4-MU-chitotrioside)
in 20 mM Na Acetate pH6, 200 mM NaCl. Reactions were incubated at 37°C for 30 minutes, free 4-Methylumbelliferone was measured at emission at 513 nm and excitation at 532 nm.
Template DNA was prepared by PCR using the Q5 High-Fidelity 2X Master Mix (NEB #M0492) (T7 promoter and terminator regions included). PCR product was purified using the Monarch PCR & DNA Cleanup Kit (5 μg) (NEB #T1030). Final concentration was adjusted to 250 ng/µl.
vGFP mRNA was prepared using NEB's HiScribe T7 Quick High Yield RNA Synthesis Kit (NEB #E2050), according to the recommended protocols, followed by purification using the Monarch RNA Cleanup Kit.
2 µl of CFPS reaction was mixed with water and Blue Loading Buffer Pack (NEB #B7703) and loaded onto 10/20% Tris-Glycine gels
(Invitrogen). The molecular weight marker used was the Unstained Protein Standard, Broad Range (10-200 kDa) (NEB #P7717).
His-tagged proteins were purified using the NEBExpress Ni Spin Columns (NEB #S1427) as directed. Eluted fractions were cleaned (to remove imidazole) using Zeba Spin Desalting Columns, and protein concentration was measured using a Nanodrop™ spectrophotometer (Thermo Fisher).
• NEBExpress Cell-free E. coli Protein Synthesis System (NEB #E5360)
• NEBExpress GamS Nuclease Inhibitor (NEB #P0774)
• PURExpress® Disulfide Bond Enhancer (NEB #E6820)
• NEBExpress Ni-NTA Magnetic Beads (NEB #S1423)
• NEBuilder® HiFi DNA Assembly Master Mix (NEB #E2621)
• Monarch® PCR & DNA Cleanup Kit (5 μg) (NEB #T1030)
• HiScribe™ T7 Quick High Yield RNA Synthesis Kit (NEB #E2050)
• Monarch RNA Cleanup Kit (NEB #T2030)
• Q5® High-Fidelity 2X Master Mix (NEB #M0492)
• Blue Protein Loading Dye (NEB #B7703)
• 2-nitrophenyl β-D-galactopyranoside (Sigma), 4-Methylumbelliferyl-β-D-N,N’,N’’-triacetylchitotrioside (Sigma)
• Zeba™ Spin Desalting Columns (Thermo Fisher)
Efficiency on different templates
Traditionally, cell-free protein synthesis systems use DNA plasmids as templates because they are readily available and easily prepared. Recently, there has been an increasing need for linear DNA templates in applications such as high throughput screening, where linear DNA can be used directly from amplification or site-directed mutagenesis. Additionally, there are situations where protein synthesis from mRNA templates is desired. For these reasons, a lysate system that can utilize plasmid DNA, linear DNA or mRNA as a template is desirable. As shown in Figure 1A, when equimolar linear DNA templates were introduced in the NEBExpress Cell-free E. coli Protein Synthesis System, the target vGFP protein was produced, albeit at a reduced yield. Yield was almost doubled by adding 1.5 μg of NEBExpress GamS Nuclease Inhibitor, which is known to stabilize linear DNA templates in in vitro protein synthesis reactions (Figure 1A and 1B). Protein yield was also enhanced by the addition of more linear DNA. With a combination of GamS and an increased amount of linear DNA, protein synthesis can be achieved at almost the same level as the plasmid DNA templates (data not shown). Figure 1C shows vGFP synthesis from increasing amounts of mRNA, in comparison with a standard reaction with plasmid DNA.
A high-performing, versatile, and robust cell-free protein synthesis system offers the ability to rapidly produce a large number of proteins for further characterization. The NEBExpress Cell-free E. coli Protein Synthesis System was developed using genetically engineered E. coli strains, an optimized reaction buffer, and stringent manufacturing practices, and is capable of synthesizing proteins as large as 230 kDa. The yield, under optimal conditions, can reach milligrams per milliliter, with protein synthesis continuing for up to 24 hours. The versatility of this system makes it ideal for a variety of applications, including high throughput protein screening and engineering as well as synthetic biology.
Proteins as large as
Yields as high as
for a variety of applications
of protein synthesis
FIGURE 1: The NEBExpress Cell-free E. coli Protein Synthesis System can efficiently synthesize proteins from plasmid DNA, linear DNA or mRNA templates
Equimolar amounts of plasmid DNA, linear DNA, and linear DNA supplemented with GamS were used in CFPS reactions. Addition of GamS to linear DNA almost doubled the vGFP protein yield.
A. Circular vs. linear DNA
B. GamS nuclease inhibitor
CFPS of guanylate kinase (GMK) and dihydrofolate reductase (DHFR) using plasmid DNA (C), linear DNA (L) and linear DNA supplemented with GamS (L+GS). Boxed region shows an increase in yield from linear DNA with the addition of GamS.
Increasing amounts of input mRNA led to increasing yields of target vGFP. Yields were compared to the yield from a CFPS reaction using 250 ng plasmid DNA.
Enhanced yields with linear DNA
Effects of target size and temperature
The ability to use one kit for synthesis of a wide range of proteins can have a big impact on experimental efficiency. The NEBExpress Cell-free E. coli Protein Synthesis System has been shown to generate high yield of proteins from a wide range of targets (16.7–230 kDa) (Figure 2A). Further, the enzymatic activity of β-galactosidase S (230 kDa) was confirmed via colorimetric assay using 2-nitrophenyl β-D-galactopyranoside as a substrate. To our knowledge, this is the largest active protein synthesized using a cell-free system.
Moreover, protein synthesis using the NEBExpress Cell-free E. coli Protein Synthesis System can be carried out at different temperatures. As shown in Figure 2B, the β-galactosidase S showed highest yield at the optimal synthesis temperature of 28°C. Similar to in vivo protein expression, the relative rate of protein translation and folding can determine how much soluble form of the protein can be obtained. These variables can be easily tuned by incubation at different temperatures in in vitro reactions.
FIGURE 2: The NEBExpress Cell-free E. coli Protein Synthesis System can generate high yields of proteins from a wide range of targets, and can be used to optimize synthesis temperature
A. CFPS of ten different targets, indicated by red dots, and a no DNA control (neg; Lane 2). Target proteins varied in size between 16.7 and 230 kDa. Synthesized proteins include (Lanes 3 to 12): β-Galactosidase from Streptococcus (βGalS), E. coli β-Galactosidase (βGal), SP6 RNA polymerase (SP6), Firefly luciferase (FLuc), 6-phospho-beta-glucosidase (BglA), citrate synthase (CitS), venus green fluorescent protein (vGFP), guanylate kinase (Gmk), dihydrofolate reductase (DHFR), calmodulin (CaM).
B. CFPS of β-Galactosidase S from Streptococcus (βGalS) using different temperatures and amounts of input plasmid DNA template. βGalS showed the highest yield using 500 ng of DNA when synthesized at 28°C.
Disulfide bond formation
Disulfide bond formation is a challenging issue for protein expression in bacteria. This can be addressed using specialized E.coli strains that have been optimized for expression of proteins with multiple disulfide bonds, such as SHuffle®. Additionally, disulfide bond enhancers can be added to in vitro protein synthesis reactions to help correct disulfide bond formation in the target proteins. Figure 3 shows that, in the absence of such enhancers, chitinase from Plasmodium that was synthesized using the NEBExpress Cell-free E. coli Protein Synthesis System displayed minimal activity. However, in the presence of PURExpress® Disulfide Bond Enhancer, there was a significant increase in the chitinase activity.
FIGURE 3: PURExpress® Disulfide Bond Enhancer increases yield of active protein in NEBExpress Cell-free E. coli Protein Synthesis reactions
CFPS of chitinase assay under standard conditions (positive control) or following addition of PURExpress Disulfide Bond Enhancer. Chitinase CFPS with enhancer produced a higher yield of active protein at both 16°C or 25°C compared with synthesis under standard conditions.
16°C for 24 hr.
25°C for 24 hr.
Sustained protein synthesis
With the NEBExpress Cell-free E. coli Protein Synthesis System it is possible to detect high yields of target protein in approximately three hours. However, given adequate aeration and agitation, the reaction can continue for more than 10 hours, producing greater than 1 mg protein/ml (Fig 4A). The longer incubation time is particularly useful when it is necessary to carry out the protein synthesis reaction at milder temperatures. As shown in Figure 4B, the synthesis of vGFP sustained over 24 hours at 25°C produced the highest yield, estimated at 3 mg/ml.
FIGURE 4: The flexibility NEBExpress Cell-free E. coli Protein Synthesis System enables extended incubations
A. CFPS of vGFP reaction at 25, 30 and 37°C. vGFP fluorescence intensity was greatest at 25°C after 24 hours.
B. End-point fluorescent values illustrate significantly higher yields with longer incubation times at low temperatures (25°C for 24 hours) than reactions at 37°C (for 16 hours).
Co-expression of proteins
It is possible to co-express multiple protein targets in a single reaction, without consideration of co-transformation and viability of the cells, which becomes limiting with in vivo expression. As shown in Figure 5, four DNA targets were added to a single reaction using the NEBExpress Cell-free E. coli Protein Synthesis System. All four targets were produced, in a clear band, although with a slightly reduced yield compared to experiments where only one plasmid is introduced. This demonstrates the potential for the NEBExpress system to make multiple proteins for the purpose of assembling a protein complex, or to introduce multiple enzymes to engineer a metabolic pathway.
FIGURE 5: The NEBExpress Cell-free E. coli Protein Synthesis System can be used for simultaneous expression of multiple targets
CFPS reactions of four different targets, expressed individually or in combination of two, three or four targets simultaneously. Specific bands are indicated with red dots. Synthesized proteins include: E. coli β-Galactosidase (βGal), 6-phospho-beta-glucosidase (BglA), venus green fluorescent protein (vGFP), dihydrofolate reductase (DHFR); negative control (neg; Lane 9). Reactions containing four simultaneously expressed targets produced four distinct bands at a slightly less yield.
Purification of the protein product
Once protein is synthesized, it is beneficial to have a rapid method for purification, in order to be able to perform further characterization. Figure 6 shows two 50 µl NEBExpress reactions, synthesizing His-tagged green fluorescent protein (vGFP) and His-tagged guanylate kinase (GMK) that were purified using NEBExpress Nickel Spin Columns following the recommended standard protocol. The final 50 µl elution had a concentration of 0.5 mg/ml, demonstrating that cell-free protein synthesis can produce enough protein for functional and structural characterization.
FIGURE 6: Purification of protein synthesized by the NEBExpress Cell-free E.coli Protein Synthesis System yields sufficient protein for downstream analysis
SDS-PAGE gel showing two 50 µl CFPS experiments, synthesizing His-tagged guanylate kinase (GMK) and His-tagged green fluorescent protein (vGFP), purified using NEBExpress Nickel Spin Columns. Lanes show samples before purification (load), unbound material (flow), unspecific binding (wash) and bound (elute) purified sample.
Cold chain shipping has long been a challenge from an environmental standpoint. Many of our products require shipment on ice or dry ice, and maintaining proper shipping temperature conditions is critical, particularly when shipping long distances or to warmer climates. Expanded polystyrene (EPS), commonly referred to as Styrofoam®, has always been the gold standard – it is light, durable, and well-known for its insulative properties.
Unfortunately, EPS is difficult to recycle and often makes its way to landfills. To address this, we have maintained a shipping box recycling program for over 40 years – customers simply use the free return label and send their shipping box back to NEB for re-use. In the meantime, we continued to look for alternative, more sustainable solutions.
We are excited to announce that beginning this Spring, NEB will be transitioning from EPS coolers to a more sustainable solution. The ClimaCell® cooler, developed by TemperPack®, maintains the cold shipping temperature requirements needed to ship NEB products, but is 100% recyclable and requires 94% less energy to manufacture.
Meet our new ClimaCell® Shipper
NEB has always placed environmental stewardship as one of our highest priorities – one of our goals has been to continuously improve our business processes in order to minimize our impact on the environment.
How is the ClimaCell cooler made?
Does the ClimaCell cooler keep product as cold as EPS coolers?
When will customers begin to receive the ClimaCell cooler?
Will NEB still be using EPS coolers?
Will NEB still maintain its shipping box recycling program?
Where can a customer go to learn more about the ClimaCell cooler?
The ClimaCell insert is paper-based, and contains thousands of air pockets to prevent heat from moving into the package. It fits tightly inside the shipping box, which is made from corrugated cardboard. Both are 100% curbside recyclable, along with other corrugated cardboard.
Customers will start receiving the new shipper in the Spring of 2020. There may be a period of time where you receive both ClimaCell and EPS coolers, until supplies are depleted.
Until we identify additional solutions, NEB will still rely on EPS coolers for a portion of our products, including those that require shipment on dry ice (e.g. competent cells), as well as larger shipments.
Yes, we will maintain the program for any EPS coolers in circulation. If you receive an EPS cooler, you are welcome to use the free return label to send your shipping box back to NEB, where we will arrange for its proper recycling.
More information on the ClimaCell cooler can be found at www.neb.com/ShippingBox
Explore the ClimaCell® cooler
Check out the layers that make up NEB's ClimaCell cooler.
The shipping box is made from corrugated cardboard and is recyclable.
ClimaCell is a paper-based insert that performs as well as EPS and is 100% recyclable.
NEB uses ChillPak gel packs from Pack Edge, Inc. Visit www.packedgeinc.com for information on recycling and disposal.
Always store your NEB product at the recommended temperature upon receipt.
Your paper packing slip will be sitting just underneath the lid of the shipping box.
ClimaCall is a paper-based insert that performs as well as EPS and is 100% recyclable.
Yes, the ClimaCell insert was specifically designed for thermal protection. It was tested under varying shipping conditions and maintains proper temperature requirements for the delivery of NEB products. Temperature stability data can be found in our Technical Note (www.neb.com/ShippingBox).
Learn more in our video.
Honoring NEB’s founder with lectures from pioneering researchers
On Wednesday November 20th, New England Biolabs hosted the Inaugural Donald G. Comb Honorary Lecture, the first in an annual series named in honor of Don Comb, NEB’s founder and first CEO. When Dr. Comb founded NEB he brought a passion for research and a vision for the company that would integrate production of reagents for molecular biology with basic research.
By Greg Lohman, Ph.D., New England Biolabs
The annual Donald G. Comb Honorary Lecture series aims to feature speakers who have pioneered basic research with substantial impact on molecular biology and related fields, especially those whose work has significant impact on problems affecting the world at large.
In addition to the foundational work of her research group, Dr. Nogales has engaged in numerous collab-orations with academic, government and industry scientists throughout her career, is well known for her energy and passion for her work, and has been a mentor to many pursuing scientific careers of their own. She was an ideal choice for our Inaugural lecture, which was held at NEB’s Ipswich campus. Dr. Nogales spent the day at NEB meeting with scientists and touring the campus.
Our first lecturer was Dr. Eva Nogales, Howard Hughes Medical Institute Investigator and Professor of Biochemistry, Biophysics and Structural Biology at the University of California, Berkeley. Dr. Nogales is a pioneer in the application of cryogenic electron microscopy (Cryo-EM) to research in molecular biology. Cryo-EM is an imaging technique that bridges the gap between molecules and cells, allowing visualization of large protein complexes too big for crystallography but too small for light microscopy. Cryo-EM enables imaging of these large complexes by rapid freezing and evaporation of a thin layer of buffered solution, capturing the proteins still folded and in their functional arrangements with their protein and nucleic acid partners. Images of these macromolecular complexes are combined to generate a three-dimensional picture of the complex. Combining Cryo-EM structures with high-resolution structures obtained by other techniques and biochemical information allows the researchers to create a dynamic picture of these complexes in action with unprecedented levels of detail.
As a result, NEB researchers have published over 1,200 publications in peer-reviewed journals, in fields ranging from biochemistry to genomics to parasitology. The annual Donald G. Comb Honorary Lecture series aims to feature speakers who have pioneered basic research with substantial impact on molecular biology and related fields, especially those whose work has significant impact on problems affecting the world at large. Further, selected speakers will embody the values of NEB, including a passion for science, innovative thinking, environmental stewardship, mentorship, genuineness and humility.
This technique allows the Nogales lab to study the macromolecular machines of the cell – such as cytoskeleton assembly and gene regulation – as whole units through direct visualization of the structure of not only individual proteins, but also dynamic multiprotein complexes in action. New insights provided by her lab have led to a deeper understanding of the large molecular machines that power the “central dogma” of DNA replication, tran-scription and translation. These studies have enabled an understanding of how these machines function and how they can fail — from how antibiotics block the cellular protein factory to how the DNA replication machinery can introduce mistakes that result in cancer or other diseases.
Dr. Nogales, Howard Hughes Medical Institute Investigator and Professor of Biochemistry, Biophysics and Structural Biology at the University of California, Berkeley
Dr. Nogales, pictured here with NEB's founder, Don Comb, also received a piece of custom artwork from local artist, Michael Updike
Structure of human transcription factor IID (TFIID), reconstructed from Cryo-EM images of the complex (top left). TFIID is a key component of the transcription pre-initiation complex, required for proper loading of RNA polymerase II onto core promoter DNA. CryoEM, in combination with crosslinking mass spectrometry and biochemical data, allowed the structures of this ~1.3 MDa transcription factor to be determined in biologically relevant conformations. Images adapted from Science. 2018 Dec 21; 362(6421): eaau8872 with permission of the authors.
Listen to Dr. Nogales discuss her work and career in our podcast
Watch Dr. Nogales' lecture from her visit to NEB's campus
Can looking to the future help preserve a historical fishery against climate change?
By Jake Kritzer, Ph.D., Environmental Defense Fund
The New England groundfish fishery
Note: This article is modified from a post in the Environmental Defense Fund’s “Fisheries for the Future” series published October 10, 2019.
New England’s storied groundfish fishery, which targets cod, haddock, a variety of flatfishes, and other bottom-dwelling predators, is among the oldest fisheries in the United States. It was once said that a fisherman could walk across the water on the backs of plentiful cod. The fishery fueled the regional economy following European settlement, and created a rich maritime heritage that continues today. That heritage first belonged to Native American peoples who long pre-dated colonization.
Preserving this iconic fishery as well as the economy and culture it has subsequently built will require looking forward to an ocean evolving under a changing climate.
Fish on the move – forward-looking manage-ment of habitat and prey
The Gulf of Maine and Georges Bank, the complex basin and shallow underwater plateau that have been the foundation of our regional fisheries, sit at the southwestern edge of the range of many North Atlantic species. Historically, these have been coldwater ecosystems due to the Labrador Current delivering frigid polar waters southward from the Arctic. However, this corner of the Northwest Atlantic is warming faster than almost anywhere else on Earth. Warming waters are causing rapid shifts in the distribution of many species, generally to the north and offshore, seeking preferred water temperatures. Thus, species we normally associate with the Mid-Atlantic — black sea bass, summer flounder, striped bass, and others — are expected to become more abundant in New England as colder water species push northward. Fishermen will see a shifting mix of species as warming progresses, and governance must also change to manage the shifting portfolios.
Climate change is making Atlantic cod recovery especially difficult, but geographical shifts might have some benefits. Cod have become concentrated in a small pocket in the western Gulf of Maine bounded by Cape Ann and Cape Cod. Elsewhere, overfishing has caused near-complete localized extinction. Although warming waters are already decreasing the productivity of cod, spreading the stock more widely across the Gulf of Maine could increase resilience relative to today’s much more restricted distribution by hedging bets against localized declines. Important efforts to restore coastal prey fishes that cod feed on, especially searun herring, are helping to give cod a chance where they have been lost.
If cod return to those areas, they will need time to re-establish. That process will be more complicated in a changing ecosystem, for the nature of seafloor habitats, water temperatures, surrounding fish and invertebrate species, and other factors will be different from what cod once knew. It was therefore with laudable foresight that the New England Fishery Management Council and National Marine Fisheries Service created a fishery closed area offshore from Penobscot Bay. That refuge is helping protect important habitats and can enable fledgling spawning groups to grow and possibly serve as a source of replenishment to areas elsewhere in the Gulf of Maine.
Cod have become concentrated in a small pocket in the western Gulf of Maine bounded by Cape Ann and Cape Cod.
Gulf of Maine
Climate change and uncertainty — responsive fishing policies for evolving conditions
Of the 20 stocks in the groundfish fishery, most live primarily away from shore. But one unique species, the winter or blackback flounder, historically moved inshore to spawning and nursery grounds in estuaries and salt ponds in the wintertime. Key habitats in those areas, including salt marshes, eelgrass beds and oyster reefs, are especially susceptible to climate change as sea levels rise, waters warm, and storms intensify. These habitat changes, among other impacts, mean that winter flounder are expected to suffer especially strong declines in productivity.
However, some winter flounder are known to spawn offshore as well. This means the stock might have the ability to counteract reduced inshore spawning success by capitalizing on deeper and colder waters. The effect of this life history diversity is but one of many scientific uncertainties we must confront, among other uncertainties related to climate change and incomplete accounting of how many fish are caught. Untangling these uncertainties and applying our findings to forward-looking management strategies will not be easy, but there are steps we can take in anticipation of changes that will come.
One unique species, the winter or blackback flounder, historically moved inshore to spawning and nursery grounds in estuaries and salt ponds in the wintertime.
A central element of any fishery management strategy is a harvest control rule, or HCR, which determines how many fish can be caught based on how many fish are in the water. An HCR is arguably where science most directly confronts policy in fisheries management, as it reveals a great deal about objectives, scientific understanding and uncertainties, and risk tolerance. In many fisheries, the HCR is simply to fish at a fixed but precautionary rate of fishing mortality that strikes a balance between achieving high yields when the stock is large, but not overfishing when the stock is smaller. However, when facing climate-driven declines in productivity that are exacerbated by scientific uncertainty, the HCR must be more responsive.
Such an approach is not yet used for New England groundfish, but could be adopted more readily than other measures.
Even if we do not understand all of the changes taking place, fishing mortality should decrease as we detect declines and can then rise again with evidence of recovery.
New England can have abundant fisheries
For those who call New England home, the groundfish fishery has sculpted our waterfronts, history, folklore and cuisine. It can remain an indelible part of our region, as long as we look to the future while we embrace the past. The ecosystem will function differently as climate change continues to unfold and we must prepare for that future. Strategic use of protected areas and responsive harvest policies, alongside other actions like recovery of prey fish and improved monitoring to track changes, can help us keep pace with a changing ocean and retain vibrant fisheries as part of our regional economy and culture.
View more blog posts from the multi-part series: Fisheries for the Future