APPLICATION NOTE

Accelerating DNA Construction to Protein Expression:
A Rapid 1-Day Workflow Using NEBridge
Golden Gate Assembly®

Sean Lund, Ph.D., Colby Stoddard, Ph.D., Matthew Norton, Ph.D., New England Biolabs , Inc.
®

INTRODUCTION
Molecular biology tools enable the custom
generation of proteins with complete control
of sequence, purification tags, secretion signals,
and other performance characteristics. Whilethe breadth of tools allows researchers to create
their desired protein, this process often involves
a low-throughput and time-consuming, multiday workflow using live cells. To overcome theselimitations, we have demonstrated a completely
in vitro workflow that combines Golden Gate
DNA Assembly, rolling circle amplification (RCA),
and cell-free protein expression (CFPE) to rapidly
screen the impact of multiple protein designs
simultaneously (Figure 1, page 2). This workflow
enables researchers to generate an array of protein
variants in as little as a single day using a basic setof custom DNA vectors or insertion fragments. It
also provides a means to assess the engineering
constraints that are unique to each protein ofinterest and allows for the rapid identificationof soluble protein.
Golden Gate Assembly utilizes Type IIS restriction
enzymes to cleave DNA in a manner that
permits scarless assembly in a modular fashion,
thereby facilitating the rapid creation of protein
variant libraries. Efficient assemblies from low

complexity (2 fragments) to high complexity
assemblies (7–50+ fragments) are supported
by NEB’s optimized NEBridge reagents andaccompanying protocols (1, 2). While Golden
Gate Assembly has been widely used for overa decade, recent computational advances (3) by
NEB have made this DNA assembly approach
more robust and user-friendly, thereby enabling
the creation of simplified workflows with ahigh probability of generating the desired DNA
construct after a single round of assembly. As
such, the workflow demonstrated here can beleveraged to generate many variants of any
desired protein with minimal customizationof DNA sequences.
While Golden Gate Assembly provides a
standardized and efficient approach for geneassembly, RCA provides an ideal method
to rapidly amplify assembled genes. RCA
is a robust and highly sensitive isothermal
amplification approach that continuously
amplifies circular DNA, resulting in a highyield without specific PCR primers. Two-hour
amplification times are achieved by employing
a new phi29 polymerase engineered by NEB
called phi29-XT. This polymerase generates
more DNA product in a shorter amount of

Rapid expression of proteins using NEBridge Golden Gate Assemblyand NEBExpress Cell-free E. coli Protein Synthesis System®

1 day
NEBridge® Golden Gate
Assembly Kit

02/24

phi29-XTRCA Kit

NEBExpress® Cell-free E. coli
Protein Synthesis System

MATERIALS• NEBridge Ligase Master Mix(NEB #M1100)• BsaI-HF v2 Type IIS restriction enzyme(NEB #R3733)®

• Q5 Hot Start High-Fidelity
2X Master Mix (NEB #M0494)®

• phi29-XT RCA Kit (NEB #E1603)• NEBExpress Cell-free E. coli Protein
Synthesis System (NEB #E5360)®

• NEBExpress Ni-NTA Magnetic Beads(NEB #S1423)• SNAP-Capture Magnetic Beads(NEB #S9145)• Monarch DNA Gel Purification Kit (5 µg)(NEB #T1020)®

time versus wild-type phi29 polymerase, while
sharing key qualities that are ideal for RCA
applications, including high processivity, strong
strand displacement activity, and high fidelity.
phi29-XT polymerase is also more thermostable,with an optimal reaction temperature of 42°C,
and has improved sensitivity, supporting
amplification down to 1 fg of DNA input (4).
This speed advantage over PCR allows faster
workflows while generating ample amounts oftemplate that are amenable to cell-free protein
expression reactions such as the NEBExpress
Cell-free E. coli Protein Synthesis System. The
NEBExpress Cell-free E. coli Protein Synthesis
System is an extract-based, transcription/translation (TXTL) system derived from E. coli
cells engineered for high in vitro synthesis
performance. It is designed to synthesize
proteins ranging in size from 17–230 kDa injust 2–4 hours under the control of T7 RNAPolymerase (5).

1This system removes the need for costly and timeconsuming steps of live-cell transformation andscreening (6). Together, the use of Golden Gate
Assembly, RCA, and cell-free protein synthesis
provides a valuable tool to maximize laboratory
productivity, especially when combined with
automation, as high-throughput screening has
become increasingly prevalent in research labs.

METHODS
Gene coding sequences were split into
three fragments using NEBridge SplitSet tool,and fragments were ordered as dsDNA fragments(gBlocks , Integrated DNA Technologies) with
Golden Gate sites appended to the 5´ and 3´ends. PCR and clean-up were carried out toensure the integrity and purity of the DNA
fragments. dsDNA fragments were resuspended
to 10 ng/µl, and 1 ng was used as a templatein a 50 µl PCR reaction using Q5 Hot Start
High-Fidelity 2X Master Mix (NEB #M0494).
Amplification specificity was verified by agarose
gel electrophoresis. The PCR amplicons werethen purified using Monarch DNA Gel Extraction
kit (NEB #T1020) and eluted into 25 µl. For
higher purity protein, users should consider using
clonal sequence-verified templates and switching
the RCA reaction with PCR amplification by Q5
High-Fidelity DNA Polymerase, followed by PCRclean-up.
®

®

PCR products (25 fmol each) and vector(25 fmol) were used in a 10 µl reaction using
BsaI-HFv2 (NEB #R3733) with NEBridge
Ligase Master Mix (NEB #M1100) according
to the online protocol. The reaction was allowed
to proceed with 30 cycles of 1 minute at 37°C
followed by 1 minute at 16°C and a final 5
minutes hold at 60°C.
Golden Gate Assembly reaction mix (1 µl) was
directly used in a 10 µl phi29-XT reaction with
0.5X LAMP fluorescent dye (NEB #B1700). The
reaction was allowed to proceed at 42°C withfluorescence monitoring on a Bio-Rad C1000
qPCR instrument every minute on the FAM
channel. After 2 hours, the reaction was heatedto 65°C for 10 minutes to heat inactivate thepolymerase. It is important to set up a control
phi29-XT amplification without template as RCA
materials can easily become contaminated.
The use of filter tips is an absolute requirement®

when setting up RCA reactions. Caution must
be exercised when handling phi29-XT reagentsand products to minimize cross-contaminationof reagents and pipettes.
In a separate reaction, 1 µl of Golden Gate
Assembly reaction mix was used in a 10 µl
phi29-XT reaction without dye in a 1.5 ml tube
and incubated on an Eppendorf Thermomixer
at 42°C, 0 rpm, for 2 hours. The temperature
was then raised to 65°C for 10 minutes.
®

FIGURE 1: Overview of Workflow

Golden Gate Assembly (GGA)®

BsaI-HF v2®

Cell-free expression was carried out using theentire phi29-XT-based amplification productin a reaction volume of 200 µl with the
NEBExpress Cell-free E. coli Protein Synthesis
System (NEB #E5360) in the same 1.5 ml
tube. The reaction was carried out at 25°C
with shaking on an Eppendorf Thermomixer
at 2000 rpm overnight (14 hours). Protein
expression can be visualized by loading 2 µl of
the reaction with 10 µl water and 6 µl 3X Blue
Protein Loading Dye (NEB #B7703S) onto an
SDS-PAGE gel. In the event your protein is not
expressed, consider digesting a portion of the
RCA product to visualize amplification quality.
Next, 50 µl of NEBExpress Ni-NTA Magnetic
Beads (NEB #S1423) were washed twicewith 200 µl 1X IMAC buffer, and the washes
removed. Cell-free expression reactions(100 µl) corresponding to the N- andC-terminally His6-tagged constructs werediluted with 100 µl 2X IMAC buffer and added
to the washed beads. The resulting mixture wasresuspended repeatedly for 30 minutes allowing
the target proteins to bind. The mixture
was applied to an external magnet and thesupernatant removed. The beads were washedtwice using resuspension with 500 µl 1X IMAC
buffer supplemented with 20 mM imidazole.
The beads were then eluted in 50 µl 1X IMAC
buffer supplemented with 500 mM Imidazole.
To affix the protein to a bead, 50 µl of SNAPCapture Magnetic Beads (NEB #S9145) werespun down and washed with 100 µl 1X IMAC
buffer. The supernatant was removed and 50 µlof the SNAP-tagged constructs were added
to the beads with 50 µl of 2X IMAC buffer.
DTT was added to 12.5 mM. The mixture was
mixed at room temperature for 30 minutes ona thermomixer before sitting overnight on the

Destination
vectors

DNA inserts

NEBridge® Golden Gate Assembly Kit

AssembledDNA

Rolling Circle Amplification (RCA)

Isothermal
amplification

phi29-XT RCA Kit
Cell-free Protein Expression (CFPE)
Cell extract
Vector®

NEBExpress Cell-free E. coli
Protein Synthesis System

DNA parts are assembled using NEBridge Golden Gate Assembly
before acting as template for a phi29-XT RCA reaction.
The amplification product can then be used in a cell-free protein
synthesis reaction from which protein can be purified. The entire
workflow is completed in vitro.

benchtop. The following day the beads werespun down and washed 3 times with 100 µl 1X
IMAC buffer with vortexing between washes.
The beads were then resuspended in 100 µl 1X
IMAC buffer.

2

DNA constructs were rapidly generated andexpressed in a 24-hour, in vitro workflow.
Assemblies were constructed using 3 insert
fragments to further demonstrate the utility
and efficiency of Golden Gate Assembly withmodular systems. The three-fragment assemblies
were constructed into expression vectors thatwere subjected immediately to RCA to generate
templates for cell-free expression. After assembly
construction, the entire workflow was conducted
on a Thermomixer providing support that suchworkflows could be scaled and automated. By
using Golden Gate Assembly with standardized
vectors, multiple assemblies could be performedin parallel using the same DNA inputs withdifferent accepting vectors. This plug-andplay methodology enables rapid screening ofvarious expression constructs without the needto order custom DNA for each expression. By
cloning multiple constructs at once, researchersinvestigating a single target or multiple targets
in a molecular foundry can rapidly construct,express, and assay their target proteins. Here
we show a rapid workflow expressing tagged
constructs with minimal perturbance to native
sequences from amplification throughprotein purification.
NEBridge Ligase Master Mix performs
high-efficiency and high-fidelity Golden
Gate Assembly with a broad assortment of
NEB Type IIS restriction enzymes. NEB
has optimized the conditions that allow for
convenient, efficient, and accurate Golden
Gate Assembly; users need only choose theirpreferred NEB Type IIS restriction enzyme andadd DNA substrates to be assembled. By using
NEBridge Ligase Master Mix, minimal ligation
sequences of 4 bp can be added to 5´ and 3´ends of inserts, thereby facilitating the use ofcommon overhangs between destination vectors
adding different functionality. If homologous
sequence cloning methods were used, adding
approximately 6 amino acids to each N- andC-terminus would be required, which could
greatly disrupt the protein sequence to
be investigated.
Four destination vectors were designed:
N-terminal His6, C-terminal His6, N-terminal
SNAP, and C-terminal SNAP (Figure 2).
The His6 tags enable the use of Ni-affinity
chromatography commonly used in protein
purification. The SNAP tags functionalize the

FIGURE 2: Vector and construct design using Golden Gate Assembly

GFP

His6 tags
Enable use of Ni-affinity
chromatography (commonlyused in protein purification)

N-His6 tag

BsaI-HF®v2 (Type IIS RE)
T7
promoterT7
terminator

Destination
vectorori

C-His6 tag
SNAP tags
Help generate animmobilized target protein(make it compatible with
SNAP-Capture beads)

N-SNAP tag
Rnka

C-SNAP tag

NEBridge Golden Gate Assembly allows for plug and play assembly of multiple vector constructs. The GFP protein is seamlessly cloned andassembled from 3 fragments into the vector containing either N- or C-terminal His tags, or N- or C-terminal SNAP tags, including the necessary
T7 promoter, RBS, T7 RNAP terminator sequences, origin of replication and antibiotic resistance marker.

target protein with a fusion that is compatible
with NEB SNAP-Capture beads to generate
an immobilized protein through a covalent
linkage in the presence of a reducing agent.
Using these two systems, we aimed to showthat this construction-to-expression workflow
is compatible with both soluble protein
purification as well as immobilized products.
Destination vectors containing an origin and an
antibiotic marker were designed to utilize thesame overhangs when treated with the TypeIIS restriction enzyme, BsaI-HFv2 (Figure 2).
The multiple cloning sites were all flanked
with a T7 promoter sequence upstream anda T7 terminator sequence downstream. The
5´ overhang was designed as “ACCA” where“A” represents the start codon of the ORF
being investigated. This tag adds an additional
threonine between any N-terminal tag andthe investigated ORF. When using destination
vectors not encoding an N-terminal tag, the“ACC” in the overhang is part of the spacer
sequence between the ribosomal binding site
and the coding sequence (CDS), generating aseamless CDS without an N-terminal addition.
The 3´ overhang is “ATTC” wherein the stopcodon is not included. The C-terminus has anisoleucine followed by a leucine (“CTG”). For
vectors generating a C-terminal tag, the tag
sequence immediately follows this isoleucineleucine (IL) linker. If the vector does not encodea C-terminal tag, a stop codon (*) follows the IL
linker sequence adding a total C-terminal linker
of: IL*.

Inserts were designed using NEBridge SplitSet
Tool to divide the desired sequence into
3 fragments. The overhangs used were thenverified with NEBridge Ligase Fidelity Viewer
with the added overhangs: “ACCA and ATTC” toensure the robustness of the assembly. Fragments
were next amplified using Q5 Hot Start HighFidelity 2X Master Mix from dsDNA fragments.
The amplified fragments were purified using theMonarch DNA Gel Extraction kit and quantified
by absorbance. Stoichiometric amounts of eachpiece and backbone were then used in a Golden
Gate Assembly reaction with BsaI-HFv2 andNEBridge Ligase Master Mix.
®

®

FIGURE 3: Rolling Circle
Amplification quickly produceshigh yield of DNA from NEBridge
Golden Gate Assembly reactions

12
10

RFU (103)

RESULTS

8
6
4
2
0
0

10

20

30

40

50

60

Time (min.)Fluorescence monitoring using phi29-XT DNA polymerase
demonstrates fast and efficient amplification of DNA template within
2 hours. Image of real-time fluorescence results shows saturation at
60 minutes or less. Black triangles are non-templated amplification.

3

FIGURE 4: GFP is highly pure following cell-free protein expression
and purification

2
El
uti
on

W
as
h

W
as
h

FT

CF

PE

1

C-terminus

W
as
h1
W
as
h2
El
uti
on

M

kDa
250
180
130

Co
ntr
ol
CF
PE
FT

N-terminus

95
72

55
43

34

Can recover both
poorly- and highlyexpressing constructs

26

17

10

SDS-PAGE showing the different His6-tagged GFP proteins that were expressed by NEBExpress Cell-free E. coli protein synthesis system andpurified using NEBExpress Ni-NTA Magnetic Beads. While the N-terminally tagged construct expressed poorly, protein was still obtained after
purification. The C-terminally tagged construct expressed well resulting in high yield of purified protein.

FIGURE 5: GFP is immobilized and functionalized on magnetic beads

NC

Settled
beads

N

C

Resuspended
by pipettingNC

10-second
centrifugeNC

30-second
centrifuge

N

Following the assembly of 3 parts into thedestination vectors, the assembly mixes werediluted into a phi29-XT amplification reaction
performed on a temperature block without
shaking, and a high yield of DNA was quickly
obtained (Figure 3, page 3). After amplificationfor 2 hours and heat inactivation at 65°C for
10 minutes, the reaction was cooled to room
temperature, and a master mix of NEBExpress
Cell-free E. coli Protein Synthesis was directly
added to the tube. The mixture was incubatedovernight with vigorous shaking. 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 using an E. coli strain
genetically engineered to maximize the stabilityof template DNA and RNA and the protein
products, a highly optimized reaction buffer, anda stringent biomanufacturing process.
The following morning, after 14 hours of
incubation, protein synthesis was verified by
SDS-PAGE. In one workflow, 100 µl of theHis6-tagged reactions were purified using 50 µl
NEBExpress Ni-NTA Magnetic Beads (Figure 4).
A stark contrast in yield was observed between
the N- and C-terminally His6-tagged proteins.
Despite the low yield of the N-terminally tagged
construct, the purified protein was isolated andobserved. The difference in yield demonstrates
the need for screening multiple constructs inparallel. In a separate workflow, the SNAP-tagged
expressed proteins were affixed to 50 µl SNAPCapture Magnetic beads in the presence of DTT(Figure 5). These two workflows demonstrated
the compatibility of the material from
NEBExpress Cell-free E. coli Protein Synthesis
with our protein purification products.

C

5-minute
settling time

SNAP-functionalized proteins were captured using SNAP-Capture Magnetic Beads. As shown, SNAP-tagged GFP is detected on the beads when
they are settled. After resuspension and pelleting, GFP fluorescence is only detected on the beads showing a strong association between theprotein of interest and bead. N = N-terminus, C = C-terminus.

4CONCLUSION

References

The workflow described herein represents a seismic shift in the time it takes to go fromcloning reaction to purified protein relative to typical cloning and subsequent protein
expression protocols. NEBridge Golden Gate Assembly reactions were successfully used as
templates for amplification by phi29-XT. The amplified material was suitable as an input forcell-free protein synthesis as shown by the demonstration of purifying His6-tagged constructs
as well as SNAP-Capture. Due to the precision of assemblies using NEBridge Golden Gate
Assembly, the rapid amplification of the phi29-XT RCA kit, and the high yield of theNEBExpress Cell-free E. coli Protein Synthesis system, genes can be cloned into expression
constructs and expressed within a day.

1. Pryor, J.M., et al. (2022) ACS Synth. Biol. 11: 2036-2042.
2. Pryor, J. M., et al. (2020) PLoS One, 15(9): e0238592.
3. Potapov, V. et al. (2018) ACS Synth. Biol., 7: 2665–2674.
4. phi29-XT RCA Kit Instruction Manual:
New England Biolabs, Inc.
5. Magnelli, P., et al. (2020) Technical Note: NEBExpress
cell-free E. coli protein synthesis system – a high performance
E. coli cell lysate-based system for in-vitro protein synthesis.
New England Biolabs, Inc.
6. Buss, J., et al. (2023) Application Note: Scaling down to scale
up – Miniaturizing cell-free protein synthesis reactions with the
Echo 525 Acoustic Liquid Handler. New England Biolabs, Inc.

Learn more about NEBridge Golden Gate Assembly at
www.neb.com/goldengate. Try our NEBridge Ligase Fidelity Tools
for Golden Gate Assembly at ligasefidelity.neb.com
For more information about the phi29-XT RCA Kit,
please visit www.neb.com/E1603
Learn more about protein expression at NEB
at www.neb.com/proteinexpression

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