bio-restriction-golden-gate-assembly

name	bio-restriction-golden-gate-assembly
description	Design and validate Type IIS scarless DNA assembly (Golden Gate, MoClo) using Biopython Bio.Restriction. Screens parts for internal BsaI/BsmBI/BbsI/SapI sites (domestication), previews the fusion overhangs a digest exposes, and validates a fusion-overhang set for distinctness and fidelity. Use when designing a Golden Gate or MoClo assembly, domesticating a part by removing internal Type IIS sites, or choosing and checking fusion overhangs for one-pot assembly.
origin	openai4s
category	bioskills/restriction-analysis
metadata	{"tool_type":"python","primary_tool":"Bio.Restriction","third_party":{"name":"GPTomics/bioSkills","repository":"https://github.com/GPTomics/bioSkills","commit":"d91ed3d563019e649dc854c56ccd62551359488a","license":"MIT"}}

Version Compatibility

Reference examples tested with: BioPython 1.83+ (API verified on 1.86)

Before using code patterns, verify installed versions match. If versions differ:

• Python: pip show biopython then help(Bio.Restriction.BsaI.search) to confirm the search API

If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.

Golden Gate / Type IIS Assembly

"Design (or check) my Golden Gate assembly" -> Make each part free of the assembly enzyme's internal sites, and give every junction a distinct, well-behaved fusion overhang, so one tube of enzyme plus ligase builds the construct directionally and scarlessly.

• Python: Bio.Restriction to find internal Type IIS sites and read the overhang a cut exposes; the overhang-set rules are sequence logic, not a database call.

The whole method rests on one property: a Type IIS enzyme cuts outside its recognition sequence, so the 4-nt overhang it leaves is set by the user's flanking DNA, and the recognition site is placed to be removed from the final product. Because the ligated junction no longer contains the site, the enzyme cannot re-cut it, so digestion and ligation run together in one pot. Two design obligations follow, and both are what this skill checks: (1) domestication -- no part may contain an internal copy of the assembly enzyme's site, or it will be fragmented during assembly; (2) a set of distinct, non-palindromic fusion overhangs -- one per junction -- so parts assemble in exactly one order.

Choosing The Assembly Enzyme

Enzyme	Recognition	Overhang	Typical role
BsaI (Eco31I)	GGTCTC(1/5)	4 nt 5'	The default Golden Gate / MoClo Level 1 enzyme; BsaI-HFv2 for fidelity
BsmBI (Esp3I)	CGTCTC(1/5)	4 nt 5'	MoClo Level 0 / Level 2 (alternates with BsaI between levels)
BbsI (BpiI)	GAAGAC(2/6)	4 nt 5'	Alternative when BsaI/BsmBI sites cannot be domesticated out
SapI (LguI)	GCTCTTC(1/4)	3 nt 5'	3-nt (codon-length) overhangs for reading-frame-preserving fusions

Hierarchical systems (MoClo, Golden Braid) alternate enzymes between levels so each assembly round removes the previous level's sites: assemble Level 0 -> 1 with one enzyme, 1 -> 2 with the other. Pick the level's enzyme first, then domesticate every part against it.

Method Context

	Golden Gate (Type IIS)	Classic restriction-ligation (Type IIP)	Gibson assembly
Junction defined by	user-designed 4-nt overhang (3 for SapI)	the enzyme's fixed overhang	~20-40 bp designed homology
Scar	none (site removed from product)	a restriction-site scar at each junction	none
Reaction	one-pot, one-step (37/16 C cycling)	sequential digest -> purify -> ligate	isothermal 50 C
Fragments per reaction	many (20-30+, more with optimized sets)	few	several
Main constraint	domestication; distinct overhang set	needs available compatible sites	terminal homology only

Choose Golden Gate when assembling several parts repeatedly from a standardized library; classic digestion for a one-off two-piece clone with convenient sites; Gibson when parts cannot be domesticated or no site layout works.

Domesticate: Find Internal Sites

Goal: Confirm a part carries no internal copy of the assembly enzyme's site (on either strand), and locate any that must be removed.

Approach: enzyme.search(seq) finds Type IIS sites on both strands (the recognition sequence is asymmetric, so its reverse-complement is detected too). Any hit inside a part is a defect to silently mutate away.

from Bio import SeqIO
from Bio.Restriction import BsaI, BsmBI, BbsI, SapI

record = SeqIO.read('part.fasta', 'fasta')

for enzyme in (BsaI, BsmBI, BbsI, SapI):
    hits = enzyme.search(record.seq)            # both strands; recognition site is asymmetric
    status = 'clean' if not hits else f'{len(hits)} internal site(s) at {hits} -> domesticate'
    print(f'{enzyme} ({enzyme.site}): {status}')

Domesticate: Break A Site By A Silent Mutation

Goal: Remove an internal site from a coding part without changing the protein.

Approach: Anchor on the recognition sequence itself (on both strands), not the cut position -- a Type IIS enzyme cuts outside its site, so mutating the codon at the cut would not touch the site. Walk the codons overlapping each recognition-site occurrence, swap one for a synonymous codon that breaks the site, and assert the protein is unchanged. This needs the reading frame.

from Bio.Data import CodonTable
from Bio.Seq import Seq

def domesticate_cds(cds, enzyme, frame=0):
    '''Remove an enzyme's internal sites from a CDS by synonymous codon swaps (reading frame `frame`).'''
    table = CodonTable.unambiguous_dna_by_id[1]
    syn = {}
    for codon, aa in table.forward_table.items():
        syn.setdefault(aa, []).append(codon)
    s = list(str(cds).upper())
    end = frame + 3 * ((len(s) - frame) // 3)
    protein = str(Seq(''.join(s[frame:end])).translate())
    site = str(enzyme.site)
    motifs = (site, str(Seq(site).reverse_complement()))    # both strands; Type IIS sites are unambiguous
    for _ in range(len(s)):
        seqstr = ''.join(s)
        if not enzyme.search(Seq(seqstr)):
            break
        hit = max(seqstr.find(m) for m in motifs)           # a recognition-site start (either strand)
        for ci in range(((hit - frame) // 3) * 3 + frame, hit + len(site), 3):
            if ci < frame or ci + 3 > len(s):
                
            codon = .join(s[ci:ci + ])
            alt = ((a  a  syn.get(table.forward_table.get(codon), ())
                         a != codon   enzyme.search(Seq(.join(s[:ci] + (a) + s[ci + :])))), )
             alt:
                s = s[:ci] + (alt) + s[ci + :]
                
     (Seq(.join(s[frame:end])).translate()) == protein   
     Seq(.join(s))

Preview The Fusion Overhangs A Digest Exposes

Goal: Read the actual 4-nt overhang each Type IIS cut would leave, to confirm junctions match as designed.

Approach: Anchor on the literal forward recognition sequence so only forward-oriented sites are read (a reverse-oriented site cuts on the other side and would otherwise return a misleading overhang). The 5' overhang starts fst5 bases after the recognition-site start.

from Bio.Restriction import BsaI

def forward_overhangs(seq, enzyme=BsaI, width=4):
    '''4-nt overhangs at FORWARD-oriented Type IIS sites, anchored on the recognition sequence.'''
    s, site, out = str(seq).upper(), str(enzyme.site), []
    i = s.find(site)
    while i >= 0:
        cut = i + enzyme.fst5                  # top-strand cut offset from the site start
        out.append(s[cut:cut + width])
        i = s.find(site, i + 1)
    return out

# Reverse-oriented sites cut on the other side; for a full construct, design the overhangs
# explicitly (below) rather than inferring every one from sequence.

Validate A Fusion-Overhang Set

Goal: Check that the overhangs chosen for all junctions assemble uniquely and ligate efficiently.

Approach: Apply the design rules: every overhang distinct; none palindromic (self-ligates); no overhang equal to the reverse complement of another (cross-ligates); avoid all-identical bases. High-throughput ligation-fidelity data (Potapov 2018) underlies curated high-fidelity sets used for large assemblies.

from Bio.Seq import Seq

def validate_overhang_set(overhangs):
    issues = []
    if len(set(overhangs)) != len(overhangs):
        issues.append('duplicate overhangs (parts assemble ambiguously)')
    for o in overhangs:
        if o == str(Seq(o).reverse_complement()):
            issues.append(f'{o} is palindromic (self-ligates)')
        if len(set(o)) == 1:
            issues.append(f'{o} is a homopolymer (low ligation fidelity)')
    rc = {o: str(Seq(o).reverse_complement()) for o in overhangs}
    for a in overhangs:
        for b in overhangs:
            if a != b and rc[a] == b:
                issues.append(f'{a} is the reverse complement of {b} (cross-ligates)')
    return issues or ['overhang set OK']

print(validate_overhang_set(['AATG', 'GCTT', 'TACT', 'GGGG']))

Common Errors

Symptom	Cause	Fix
Assembly drops or scrambles a part	An internal Type IIS site fragmented it	Domesticate every part against the level's enzyme before assembly
Junctions ligate in the wrong order or orientation	Two junctions share an overhang, or one is the reverse complement of another	Use a distinct, non-self-complementary overhang per junction; check with validate_overhang_set
Empty or low-efficiency assembly	Palindromic or homopolymer overhang, or wrong enzyme/buffer cycling	Avoid palindromic/homopolymer overhangs; cycle 37/16 C with a Type IIS enzyme + T4 ligase
search() misses a reverse-oriented site	Site too close to the sequence end so the cut falls off it	Domesticate on the full part in context, not a trimmed fragment
Recognition site still present in the product	Site placed so the cut does not remove it	Orient Type IIS sites so cleavage excises them from the assembled junction

Related Skills

• enzyme-selection - Choose a classic restriction enzyme when scarless assembly is not needed
• restriction-sites - Find any enzyme's sites in a part
• fragment-analysis - Predict fragments to verify an assembly digest
• genome-engineering/grna-design - Design constructs that this assembly will build
• sequence-manipulation/transcription-translation - Confirm domestication kept the reading frame

References

• Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability. PLoS One. 2008;3(11):e3647. doi:10.1371/journal.pone.0003647
• Engler C, Gruetzner R, Kandzia R, Marillonnet S. Golden gate shuffling: a one-pot DNA shuffling method based on type IIs restriction enzymes. PLoS One. 2009;4(5):e5553. doi:10.1371/journal.pone.0005553
• Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S. A modular cloning system for standardized assembly of multigene constructs. PLoS One. 2011;6(2):e16765. doi:10.1371/journal.pone.0016765
• Potapov V, Ong JL, Kucera RB, et al. Comprehensive profiling of four base overhang ligation fidelity by T4 DNA ligase and application to DNA assembly. ACS Synth Biol. 2018;7(11):2665-2674. doi:10.1021/acssynbio.8b00333