L3 ITERATIVE CLONING KEY POINTS PART 1

Differences between Type II and Type IIs enzymes

1. Type II recognize and cut in palindromic sequences, Type IIs do not 

2. Type II can re-ligate with compatible ends only 

3. Type IIs ends are programmable and can re-ligate with complementary ends, 

allowing the creation of a grammar of compatible ends: 256 different overlaps can 

be made with 4 bp (44). Grammar allows fragment design for reusable combinations: 

promoters are always upstream to ORFs that are always upstream to 3’UTRs 

Golden Gate cloning 

Type IIs enzyme (Bsa1) is combined with T4 ligase in 1 reaction with parts vectors with compatible ends to each other and to the destination vector 

Advantages of GOLDEN GATE

1. One pot cloning  

2. Small scars (4 bp overhangs)  

3. Multi fragment assembly based on fixed grammar  

4. Reusable parts that can be combined in many ways 

Disadvantages of GOLDEN GATE

1. Type IIs enzymes used for cloning need to be removed from all the parts that are used  

2. Limited complexity 

Golden Braid cloning 

1. Uses 3 vectors: parts vectors (pUPD), alpha (A vectors) and omega (O vectors) 

2. Parts vectors are assembled into A vectors. Two compatible A vectors (A1 + A2) go  into O vectors. Two compatible O vectors (O1 + O2) go into A vectors. Rounds of A to O and O to A keep growing the construct into ever complex designs 

3. All vectors have two Type IIs enzymes that face each other: BsaI (GGTCTC) and BsmBI (CGTCTC) 

Advantages of Golden Braid  

1. Assemblies can be added to each other by going back and forth between alpha and omega level vectors, dramatically increasing the complexity  

2. Pieces are reusable making assemblies of complex constructs easy and modular 

pUPD vectors: universal part domesticator 

Used to domesticate parts

Multiple parts are assembled into A vectors to make transcription units (promoter + ORF + 3’UTR) 

The setup of golden braid pUPD vectors

BsaI outside and BsmBI inside on the LacZ’ fragment

What kind of resistance can pUPD vectors have?

Ampicillin resistant or Chloramphenicol resistant

A vectors: alpha vectors 

1. To make the first set of Transcription Units (TUs = promoter + ORF + 3’UTR) or other assemblies 

2. BsmBI outside and BsaI inside on the LacZ’ fragment 

3. Kanamycin resistant 

4. Use GGAG and CGCT grammar on 5’ and 3’ end respectively for entry 

Difference between A1 and A2 vectors

A1 vectors: BsmBI and BsaI are 6 bp apart on 5’ end and 10 bp on the 3’ end 

A2 vectors: same as A1 but 5’ and 3’ spacing is flipped 

O vectors: omega vectors 

1. To make the first set of Tus (promoter + ORF + 3’UTR) 

2. BsaI outside and BsmBI inside on the LacZ’ fragment 

3. Chloramphenicol resistant 

4. Use GGAG and CGCT grammar on 5’ and 3’ end respectively for entry 

Omega vectors are two alpha vectors combined

Difference between O1 and O2 vectors

O1 vectors: BsaI and BsmBI are 6 bp apart on 5’ end and 10 bp on the 3’ end 

O2 vectors: same as O1 but 5’ and 3’ spacing is flipped 

BASIC GRAMMAR RULES

1. Promotor: GGAG-AATG 

2. ORF: AATG-GCTT 

3. 3’UTR: GCTT-CGCT 

4. Promotor + ORF + 3’UTR: GGAG-AATG + AATG-GCTT + GCTT-CGCT together go into A 

as GGAG-CGCT beginning to end 

5. Additional grammars exist to make more complex combinations into A vectors 

Domestication 

Domestication is done through synthesis or PCR by adding a BsmBI recognition site 

to the PCR primers followed by 1 random base, the CTCG overhang for entry into 

pUPD and the 4bp grammar for future assembly in A 

What are the two reasons for doing domestication?

1. Remove internal type IIs sites

2. Create 4bp overhangs (grammar)

What does a primer typically look like in domestication?

CGCGCGTCTCNCTCGNNNN

The breakdown of the parts of this primer: CGCGCGTCTCNCTCGNNNN

a. CGCG: extra GC rich bases for the Type IIs enzyme to efficiently cut 

b. CGTCTC: BsmBI recognition site 

c. CTCG: pUPD compatible overhang 

d. NNNN: grammar for future assembly in A 

What is the Golden Braid reaction?

1. 1 pot reaction with Type IIs enzyme, T4 ligase and parts at precise concentrations 

(40 ng for parts and 75 ng of destination vector) 

2. Reaction in set up in a thermocycler that goes between digestion and ligation cycles 

at 37°C and 16°C respectively, repeated 25 to 5 times followed by final enzyme 

inactivation step for 20 min at 85°C 

3. 1/10 of the reaction is transformed into DH10B bacteria 

4. After 1 hr recovery of the transformation at 37°C, 1/10 of the cell volume is plated on 

LB agar with the appropriate antibiotic 

5. White colonies are verified by colony PCR, subsequently prepped, fingerprinted and 

sequence verified 

pUPD domestication step reactions

1. Design primers – Amplify your gene/fragment of interest with primers that add type restriction enzyme recognition sites (usually BsaI for pUPD domestication). These also include overhangs compatible with the Universal Entry Vector (pUPD). 

2. PCR amplification – Generate the fragment with the new flanking sites. 

3. Golden Gate reaction (domestication) – Mix together: 

• PCR product (your gene with flanking BsaI sites) 

• pUPD vector (entry vector for TU grammar) 

• BsaI enzyme (cuts outside recognition sequence, exposing designed overhangs) 

• T4 DNA ligase (seals compatible overhangs) 

→ BsaI cuts away any internal restriction site conflicts, producing “domesticated” parts. 

4. Ligation into pUPD – Your fragment is inserted into the Universal Entry Vector in the correct orientation and free of type IIS conflicts. 

5. Transformation & verification – Introduce into E. coli, then screen colonies (colony PCR, sequencing) to confirm the domesticated part. 

Vector Configurations

1. pUPD: Bsa1 - Bsmb1 (Amp to Kan)

2. A vectors: BsmB1 - Bsa1 (Kan to Cam)

3. O vectors: Bsa1 - BsmB1 (Cam to Kan)