TITLE: Golden Gate Assembly
AUTHORS: Isaac  Núñez, Tamara  Matute, Fernan  Federici

[DESCRIPTION]
The Golden Gate technique allows the assembly of genetic sequences from libraries of standardized basic components, which are cleaved from their donor vectors and concatenated in the acceptor vector in a defined order. This reaction is subsequently transformed into competent bacterial cells that are grown overnight to reveal positive colonies carrying the correctly assembled vector. 

The cleavage of the components is done by a TypeIIS restriction enzyme, which recognizes asymmetric DNA sequences and cleaves outside of this site, leaving overhangs in a directional way (Figure 1A). This property allows the creation of ordered assembly positions by defining synthetic syntaxes of these overhangs (Figure 1B).

  
Within the different methodologies and libraries based on Golden Gate, some methods, such as uLoop Assembly (Pollak et al, 2020), allow the cyclic assembly of increasingly large and complex vectors in which the products of a reaction are the substrates of the next level reactions (Figure 2). By this means it is possible to build libraries of easily reusable components to perform assemblies in a combinatorial way of odd and even assembly levels.

  
The standardized DNA components have to be flanked by the enzyme recognition sites and the proper syntax according to the desired position of assembly. These DNA pieces can be obtained from previously created libraries or custom made.

The reaction operates cycling between 37°C and 16°C. At the first temperature, the restriction enzyme cuts the DNA pieces. These pieces are concatenated by their overhangs sequence homology and sealed by the catalysis of a ligase enzyme at the second temperature. Once a piece is correctly assembled it cannot be cutted again, and cycle by cycle, the pieces are concatenated in the final assembly product.




[STEPS]
SECTION: Preparation of the DNA components
1. Prepare plasmids stock solutions of the desired components to be used. 
 



SECTION: Preparation of the DNA components
2. Measure the concentration of purified plasmids. 
 


SECTION: Preparation of the DNA components
3. Perform dilutions of the plasmids to working concentrations. 
It is:
15 fmol/μL for donor vectors
7.5 fmol/μL for receiver vectors

Molar concentration can be computed from mass concentration accord the next equation:

  

Then, dilutions should be made in molecular grade H2O using the standard equation:
   
 



SECTION: Golden Gate Reaction
5. Prepare the 2X Reaction Master Mix (BsaI or SapI) on ice according to the next table:
 

 

SECTION: Golden Gate Reaction
6. Mix the 5 µL of the DNA mix (step 4) with 5 µL of the 2X Reaction Master Mix (step 5). 
 

SECTION: Golden Gate Reaction
4. Prepare the DNA mix. In a pcr tube, mix 1 µLof each component to be assembled (donor vectors) and 1 µL of the acceptor vector.
 

SECTION: Golden Gate Reaction
7. Bring the tubes to a thermocycler and run it according the next program:
  
 

SECTION: Transformation and colony selection
8. Directly transform 5 µLof the reaction into 50 µL chemo-competent cells.
SECTION: Transformation and colony selection
9. Plate the transformant cells into plates supplemented with the proper antibiotic plus X-Gal 40 μg/mL and growth overnight at 37 °C

SECTION: Transformation and colony selection
10. Identify the colonies carrying the proper assembled vectors based on blue - white screening of the grown colonies (blue colonies carry intact acceptor vector and should not be selected), plus any other particular screening criteria related to your assembly (i.e. fluorescence expression, colony PCR, sequencing, etc).
 

SECTION: Golden Gate Reaction
6. Mix the 5 µL of the DNA mix (step 4) with 5 µL of the 2X Reaction Master Mix (step 5). 
 

SECTION: Golden Gate Reaction
7. Bring the tubes to a thermocycler and run it according the next program:
  
 

SECTION: Transformation and colony selection
8. Directly transform 5 µLof the reaction into 50 µL chemo-competent cells.
SECTION: Transformation and colony selection
9. Plate the transformant cells into plates supplemented with the proper antibiotic plus X-Gal 40 μg/mL and growth overnight at 37 °C

SECTION: Transformation and colony selection
10. Identify the colonies carrying the proper assembled vectors based on blue - white screening of the grown colonies (blue colonies carry intact acceptor vector and should not be selected), plus any other particular screening criteria related to your assembly (i.e. fluorescence expression, colony PCR, sequencing, etc).