ProtocolsIVTT Pipeline

Template Generation: gBlocks, Codon Optimization, Universal PCR

Standard operating procedure from the WeaveSeq experiments team, shared across the IVTT pipeline runs.

Template Generation: gBlocks, Codon Optimization, Universal PCR

Objective

Produce one IVTT-ready linear dsDNA template per binder for the top ~96 in-silico candidates of a campaign: E. coli-codon-optimized, carrying a T7 promoter, Shine-Dalgarno (SD) site, Kozak sequence, N-terminal FLAG tag, double stop, and T7 terminator. Templates are ordered as individual IDT gBlocks and amplified with one universal primer pair. They feed SOP 04 (IVTT Expression); the FLAG tag is the handle for the HTRF donor arm of the sandwich in SOP 05 (TR-FRET Plate Assay).

1. Template Strategy

Order 96 individual IDT gBlocks, one per binder, plus 2-3 control fragments (Section 6). One tube per binder means every PCR well receives a single sequence, so no pool deconvolution step is required, and any fragment that fails QC can be re-ordered on its own.

1.1 Why Amplify the gBlocks

gBlocks arrive at 250-1,000 ng each, enough for dozens of 10 µL mini-reactions directly. A single universal-primer amplification is still worthwhile because it:

banks a master stock, so repeat campaigns need only PCR reagents rather than new DNA synthesis;
produces the exact linear-template format NEB documents for PURExpress: T7 promoter, RBS, start and stop codons, terminator, and at least 6 bp after the stop;
yields a sequencing amplicon for Sanger QC.

2. Codon Requirements: E. coli Optimization and Rare Codons

Codon optimization must target E. coli. A CDS optimized against another host's codon table can carry Arg codons that are rare in E. coli, and the FLAG readout used here is unusually sensitive to them.

AGA and AGG are the rarest Arg codons in E. coli, about 5-8% of Arg codons (Gurvich 2005; Kazusa K-12). AGA/AGG, together with AUA, CCC, and CGG, severely depress expression in E. coli: under 5% of control yield in E. coli cells (Chumpolkulwong et al. 2006). Cell-free yields are appreciably higher than in cells, so the exact IVTT penalty is not quantified, but the reduction is well documented.
Tandem AGG-AGG can induce up to 50% +1 ribosomal frameshifting in highly expressed mRNAs (Spanjaard & van Duin 1988); 1.5-11% in native contexts (Gurvich 2005).
The PURE system is tRNA-limited (Li et al. 2017; Doerr et al. 2021), so rare-codon penalties are expected to be at least as severe as in cells.

Why this is a false-negative trap. Detection is N-terminal FLAG, so a premature stop or a frameshift anywhere in the CDS still reports a signal: the truncated protein fails to bind and masquerades as a non-binder. Check each binder sequence for AGA codons before ordering.

Re-optimizing the CDS with the E. coli MFC table (Section 3.2) produces zero rare codons.

3. Construct Design

Fragment layout, 5′ to 3′. Mean 320 bp, range 273-360 bp for the 51-80 AA binders this protocol targets. Fixed flanks are 61 nt at the 5′ end and about 59-60 nt at the 3′ end.

5'-TAATACGACTCACTATAGGG  GG  AGGAGG  GCCACC  ATG  GACTACAAAGACGATGACGACAAG  <binder CDS>  TAATAG  GTCGAC  TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG -3'
   └─ T7 promoter (20 nt) ┘ └SD┘  └Kozak┘ └start┘└─ FLAG = DYKDDDDK (24 nt) ──┘ └─ E. coli MFC ─┘ └2×stop┘ └6 bp─┘ └───── T7 terminator (47 nt) ─────┘

The expressed ORF is M + DYKDDDDK + binder amino-acid sequence: an N-terminal FLAG fusion whose detection handle is the anti-FLAG M2 donor arm of the HTRF sandwich (SOP 05). The CDS is back-translated from the binder amino-acid sequence with the E. coli most-frequent-codon (MFC) table (Section 3.2).

3.1 Design Rules (NEB PURExpress E6800 manual)

Element	Requirement	This design
T7 promoter	20-100 nt upstream of the CDS	TAATACGACTCACTATAGGG (20 nt), immediately 5′ of the GG spacer
Ribosome binding site (SD) and Kozak context	SD about 6-8 nt upstream of the ATG; Kozak context not required by E. coli systems	AGGAGG followed by GCCACC (6 nt), placing the SD 3′ end 6 nt from the start codon
Start / stop codons	Required	ATG, then TAATAG (TAA TAG tandem)
Post-stop spacer	At least 6 bp downstream of the stop codon (PCR templates)	GTCGAC (6 bp)
T7 terminator	Recommended for PCR templates	47-nt canonical terminator (Section 3.3)
Template amount	Starting concentration 250 ng per 25 µL reaction; valid range 25-1,000 ng	SOP 04 scales this to 10 µL mini-reactions

3.2 E. coli MFC Codon Table

AA	Codon	AA	Codon	AA	Codon	AA	Codon
A	GCG	C	TGC	G	GGC	P	CCG
R	CGT	Q	CAG	H	CAT	S	AGC
N	AAC	E	GAA	I	ATT	T	ACC
D	GAT	K	AAA	L	CTG	V	GTG
F	TTT	M	ATG	W	TGG	Y	TAT

Re-optimization with this table produces zero rare codons. The accompanying codon scan excludes AGG, AGA, CGG, CGA, ATA, CTA, CCC, GGA, and TTA. Chumpolkulwong et al. 2006 measured five of these (AGA, AGG, ATA, CCC, CGG) as severely depressing E. coli cell-free expression; the remaining four depend on minor tRNAs and are excluded conservatively. The MFC table never emits any of them.

Table basis. The table matches the Kazusa E. coli K-12 (MG1655) whole-genome most-frequent-codon table on 19 of 20 amino acids. For Arg, CGC and CGT are near-tied genome-wide (CGC 0.40 vs CGT 0.38), but CGT is the clear winner in the Hénaut & Danchin (1996) class II table for highly expressed genes (64.25 vs 32.97), so R = CGT is used. PUREfrex supplies an E. coli tRNA mixture, so templates should match E. coli codon frequency (GeneFrontier FAQ Q12).

3.3 Fixed Element Sequences (Exact)

Element	Sequence (5′ → 3′)	Notes
T7 promoter	TAATACGACTCACTATAGGG	Doubles as the universal forward primer
GG spacer	GG	2 nt; keeps the T7 +1 transcript start clean
SD	AGGAGG
Kozak	GCCACC
Start	ATG
FLAG	GACTACAAAGACGATGACGACAAG	Canonical FLAG epitope DYKDDDDK; no dibasic OmpT cleavage site
Double stop	TAATAG
3′ spacer	GTCGAC	6 bp
T7 terminator	TAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTG	47 nt
Universal R primer	CAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTA	Reverse complement of the terminator
Optional c-Myc	GAACAAAAGCTTATTTCTGAAGAGGACTTG	EQKLISEEDL (30 bp), inserted between CDS and TAATAG as a second detection tag

Because all 96 fragments plus controls share identical 5′ and 3′ flanks by construction, one universal primer pair amplifies the entire panel.

4. Materials

Item	Vendor	Catalog #	Notes
gBlocks gene fragments, 125-3,000 bp (tubes)	IDT	order item	250-1,000 ng per fragment; 2-4 business days up to 750 bp (5-8 BD above 1,000 bp); median error rate better than 1:5,000 bp; plate format has a 24-fragment minimum, so order tubes
Gene Fragments, 0.3-0.5 kb	Twist Bioscience	order item	Backup vendor for re-orders; express 2-4 BD; error rate 1:7,500
Gene Fragments, 200-500 bp	GenScript	order item	Second fallback; 5-7 BD
Standard 25 nmole DNA oligos (universal F/R pair)	IDT	order item	One primer pair covers the whole panel
Q5 2× Master Mix	NEB	confirm with vendor	High-fidelity PCR enzyme for universal amplification
DNA Clean & Concentrator-5	Zymo	confirm with vendor	Post-PCR cleanup
Qubit dsDNA HS Assay Kit	Thermo Fisher	confirm with vendor	Template quantification
Sanger sequencing	core facility	n/a	Sequencing of the top 12-20 templates
DNA LoBind tubes, 1.5 mL	Eppendorf	022431021	For fragment and stock dilutions
UltraPure DNase/RNase-free water	Thermo Fisher	10977015	Resuspension and elution
96-well PCR plate, full skirt, 0.2 mL	Corning (Axygen)	PCR-96-FS-C	Universal PCR in plate format (or Eppendorf twin.tec 0030128648)
MicroAmp Clear Adhesive Film	Thermo Fisher	4306311	Plate sealing
Aluminum foil seals for PCR	VWR	60941-126	Alternative sealing

5. Step-by-Step Protocol

5.1 In Silico QC Before Ordering (Mandatory)

Run these checks on the designed fragment set before the order is placed.

Check	Method	Gate
Reading frame	Translate all 96 fragments in silico	0/96 frame or premature-stop errors; ORF = M-DYKDDDDK-binder-STOP
FLAG present	Sequence scan	96/96 at the N-terminus, immediately after ATG
Double stop	Sequence scan	96/96 TAATAG before the 3′ spacer
Rare codons	Scan AGG/AGA/CGG/CGA/ATA/CTA/CCC/GGA/TTA	0 hits
Internal motifs	Scan CDS for T7 promoter/terminator runs	0 hits
Length sanity	Expected range for 51-80 AA binders	273-360 bp

5.2 Order the Fragments

Order 96 binder gBlocks plus 2-3 control gBlocks (Section 6) from IDT in tubes format, 250-1,000 ng each. The plate format has a 24-fragment minimum.
Keep a backup vendor for re-orders: Twist (0.3-0.5 kb, express 2-4 business days, error rate 1:7,500) or GenScript (5-7 business days).
Plan for synthesis errors. At 1:5,000 per bp, a ~320 bp fragment has about a 6% chance of at least one error, so expect roughly 6 of 96 fragments to carry one. Single-point screen wells tolerate this; dose-response binders must not (Section 5.6).

5.3 Resuspend the Fragments

Briefly centrifuge each tube (10 s) to collect the pellet.
Resuspend in 10 mM Tris pH 8.0 (IDTE) to ~10 ng/µL.
Pipette up and down 10 times; if a visible pellet remains, heat to 55°C for 5 min, vortex, spin down; equilibrate 30 min at room temperature.
Store at −20°C in DNA LoBind tubes; aliquot to avoid more than 3 freeze-thaw cycles.

5.4 Universal PCR Amplification

One primer pair amplifies all 96 fragments plus controls. Reaction (50 µL, per fragment):

Component	Volume
Q5 2× Master Mix	25 µL
10 µM universal F primer (T7: TAATACGACTCACTATAGGG)	2.5 µL
10 µM universal R primer (reverse complement of the terminator, Section 3.3)	2.5 µL
gBlock (1-10 ng)	X µL
Nuclease-free water	to 50 µL

Thermocycler program:

Step	Temp	Time	Cycles
Initial denaturation	98°C	30 s	1
Denaturation	98°C	10 s
Annealing	60°C	15 s	12-15
Extension	72°C	30 s
Final extension	72°C	2 min	1
Hold	4°C	∞

Use the minimum cycle count that gives robust yield, no more than 15: every extra cycle accumulates polymerase errors. Run in a 96-well PCR plate with 96 fragments plus 2 controls plus 1 no-template well per plate.

5.5 Cleanup, Quantification, Normalization

Clean up with Zymo DNA Clean & Concentrator-5; elute in 20 µL nuclease-free water.
Quantify by Qubit dsDNA HS; optionally check A₂₆₀/A₂₈₀ = 1.8-2.0.
Normalize to a common working concentration and bank master stocks (−20°C, DNA LoBind tubes, no more than 3 freeze-thaw cycles). The IVTT dosing target is 250 ng per 25 µL reaction (25-1,000 ng range); SOP 04 scales this to 10 µL mini-reactions (equivalent to 10 ng/µL in reaction).

5.6 Sanger QC

Sequence the top ~12-20 candidates (the dose-response set plus any screen outliers) using the universal primers.
Gate: exact match to the designed sequence. Any mismatch means re-ordering that fragment from the backup vendor (Twist), or removing the binder from the titration set and recording the exclusion.
Optional full-panel QC: sequence all 96 templates with the same primers.

6. Control Templates

Order as 2-3 extra gBlocks with the same flank design:

Control	Content	~Size	Purpose
POS1	FLAG + core streptavidin CDS	~540 bp	Binds the biotinylated target directly (core streptavidin retains biotin binding, Kd ~10⁻⁷ M; Sano & Cantor, PNAS 1995), giving maximal HTRF sandwich signal. Validates expression, FLAG detection, and the full FRET geometry end to end
CTRL	FLAG + eGFP CDS	~870 bp	Expression and donor-arm control; negative control for target binding
(spare)	duplicate POS1	~540 bp	Re-order insurance for the most-used control

Assay-level negative wells (no-template lysate, FLAG-eGFP + biotinylated target, free FLAG peptide DYKDDDDK competition at 100 µg/mL, and biotin-BSA acceptor-only) are plate controls defined in SOP 05.

7. QC Checkpoints

Stage	Method	Gate
Before ordering	In silico translation of all 96 (Section 5.1)	0 frame or stop errors; FLAG and TAATAG present; 0 rare codons
Order manifest	Compare the vendor order confirmation against the designed sequences	99/99 sequences match
Post-PCR	Qubit dsDNA HS	Stock sufficient for 250 ng/25 µL IVTT dosing per binder
Post-PCR	Agarose gel, 1.5-2%	Single band at the expected size, ±10%
Sanger (top ~12-20)	Core facility	Exact match to design, otherwise re-order or exclude
Storage	−20°C, LoBind tubes	No more than 3 freeze-thaw cycles

8. Turnaround

Milestone	Day
Order gBlocks and control fragments	0
Fragments arrive (2-4 business days, up to 750 bp tier)	2-4
Day 1: universal PCR, cleanup, quantification, Sanger submitted (top 12-20)	arrival + 1
Day 2: 96-well IVTT and single-point TR-FRET screen (SOP 04, SOP 05)	arrival + 2
Day 3: re-express top 12-20 (25 µL) and run 12-point Kd titrations, with the Sanger veto in hand	arrival + 3
Day 4: Kd fits and results recorded	arrival + 4

End to end, sequence to answer is about 6-8 calendar days (fragments 2-4 business days plus the 4-day lane); plan 8-10 with contingencies. Sanger results land on Day 2-3: use them to veto any erroneous dose-response template before the Day-3 re-expression.

Optional pilot. Order 3-4 binder gBlocks plus POS1 and CTRL first and validate them end to end through SOPs 04-05 before committing to the full 96 (adds about 2-3 days). Recommended for the first campaign; skippable thereafter.

9. Troubleshooting

Symptom	Likely Cause	Action
No amplicon for an individual fragment	Synthesis error (about 6% per fragment at 1:5,000 per bp) or lost pellet	Verify resuspension (spin, 55°C); re-order from the backup vendor; mark the binder as excluded on template QC
Multiple bands on gel	Primer mis-priming on a GC-rich CDS	Raise annealing to 62-65°C; reduce to 12 cycles; gel-purify the correct band
Low expression across the whole panel (SOP 04 QC)	Construct design error	Re-run the Section 5.1 checks; confirm FLAG is in frame and the codon table matches Section 3.2
Truncated products on anti-FLAG QC blot	Frameshift from a synthesis error	Sanger that binder; re-order or drop it
A₂₆₀/A₂₈₀ outside 1.8-2.0	Cleanup carryover	Re-clean with DNA Clean & Concentrator-5; blank against the elution buffer
CTRL (FLAG-eGFP) expresses but most binders do not	Residual rare codons or strong secondary structure in the CDS	Re-run the rare-codon scan; redesign the problem CDSs with the MFC table or switch to the Twist sequence

10. References

NEB PURExpress E6800 manual. template requirements: T7 promoter 20-100 nt upstream, RBS ~6-8 nt upstream of ATG, start/stop codons, at least 6 bp spacer after the stop, T7 terminator recommended; 250 ng/25 µL (25-1,000 ng range). Manual PDF
NEB PURExpress protocol. 37°C, 2 h. Protocol
Chumpolkulwong et al. (2006). AGA/AGG (with AUA/CCC/CGG) severely depress E. coli expression. J Struct Funct Genomics 7:31-36. PMID 16703415
Spanjaard & van Duin (1988). tandem AGG-AGG induces up to 50% +1 frameshifting. PNAS. Europe PMC PMC282334
Gurvich et al. (2005). E. coli AGA/AGG usage and expression penalties in native contexts. J Bacteriol 187:4023-4032. PMID 15937165
Kazusa codon usage database. E. coli K-12 tables. kazusa.or.jp/codon
IDT gBlocks product page. 125-3,000 bp, 2-8 business days, 250-1,000 ng, error better than 1:5,000. Page
Twist Bioscience gene fragments. 0.3-0.5 kb, error 1:7,500. Page
Sano & Cantor (1995), PNAS. core streptavidin retains biotin binding (Kd ~10⁻⁷ M); basis of the POS1 control design.
Thermo Fisher FLAG tag antibodies page. canonical FLAG epitope DYKDDDDK. Page
GeneFrontier PUREfrex FAQ Q12. PUREfrex uses an E. coli tRNA mixture whose tRNA levels mirror E. coli; template codons should match E. coli codon frequency. Q12
NEB PURExpress FAQ. rare codons or a skewed amino-acid composition can make full-length product a minor species; supplementing the "missing" tRNA may help. FAQ
Li et al. (2017). PURE is tRNA-limited: replenishing tRNAs and other substrates raised luciferase yield ~1.5-2×. Translation 5(1):e1327006.

AA	Codon	AA	Codon	AA	Codon	AA	Codon
A	GCG	C	TGC	G	GGC	P	CCG
R	CGT	Q	CAG	H	CAT	S	AGC
N	AAC	E	GAA	I	ATT	T	ACC
D	GAT	K	AAA	L	CTG	V	GTG
F	TTT	M	ATG	W	TGG	Y	TAT

AA	Codon	AA	Codon	AA	Codon	AA	Codon
A	GCG	C	TGC	G	GGC	P	CCG
R	CGT	Q	CAG	H	CAT	S	AGC
N	AAC	E	GAA	I	ATT	T	ACC
D	GAT	K	AAA	L	CTG	V	GTG
F	TTT	M	ATG	W	TGG	Y	TAT

AA	Codon	AA	Codon	AA	Codon	AA	Codon
A	GCG	C	TGC	G	GGC	P	CCG
R	CGT	Q	CAG	H	CAT	S	AGC
N	AAC	E	GAA	I	ATT	T	ACC
D	GAT	K	AAA	L	CTG	V	GTG
F	TTT	M	ATG	W	TGG	Y	TAT