52-Fragment Golden Gate Assembly using BsaI-HF®v2 (NEB #E1601)


Overview

Golden Gate Assembly of 52 fragments using BsaI-HFv2 restriction enzyme with T4 DNA Ligase and subsequent transformation into NEB 10-beta Competent E. coli (High Efficiency). 

Materials

  • NEB Golden Gate Assembly Kit (BsaI-HFv2) (NEB #E1601)
  • pGGAselect Destination Plasmid*
  • NEB 10-beta Competent E. coli (NEB #C3019)
  • NEB 10-beta/Stable Outgrowth Medium (NEB #B9035)
  • LB Agar plates with chloramphenicol
    *included in NEB® Golden Gate Assembly Kit (BsaI-HF®v2) (NEB #E1601)

Reaction set-up

Set up 20 µl assembly reactions as follows:

REAGENT ASSEMBLY REACTION
pGGAselect  Destination Plasmid(1)* 1 μl
Amplicon Inserts(2) 3 nM(3) each DNA fragment (final concentration)
T4 DNA Ligase Buffer (NEB #B0202) (10X) 2 μl
NEB Golden Gate Enzyme Kit (BsaI-HFv2) (NEB #E1601) 2 μl
Nuclease-free H2O (NEB #B1500) to 20 μl(4)

*or user provided.


(1) Destination vector must possess BsaI restriction sites at both ends of the insert sequence and in the proper orientation.
(2) Amplicon inserts must possess 5 ́ flanking bases (6 recommended) and BsaI restriction sites at both ends of the amplicon and
in the proper orientation.
(3) The NEBiocalculator® Tool (nebiocalculator.neb.com) can be used for molarity calculations.
(4) In our hands, consistent results can be obtained with final reaction volumes ranging from 5-25 μl given that the reaction buffer
and Golden Gate Enzyme Mix is scaled proportionately.

Reaction assembly protocol

SUGGESTED ASSEMBLY PROTOCOL
37°C, 48 h(5) → 60°C, 5 min→ 4°C(6)

(5) Incubation time can be modified; shorter incubation times may reduce assembly yield and additional incubation time may
improve assembly yield.
(6) Cool reaction to 4°C prior to transformation, or store completed assembly reactions at -20°C. 

Transformation

  1. For each assembly, thaw a 50 µl tube of NEB 10-beta competent E. coli cells on ice for 5–10 min.
  2. Add 2 µl of the assembly reaction; gently mix by flicking the tube 4-5 times.
  3. Incubate on ice for 30 min.
  4. Heat shock at 42°C for 30 sec.
  5. Place back on ice for 5 min.
  6. Add 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium (NEB #B9035). Incubate at 37°C for 60 min., shaking vigorously (250 rpm) or using a rotation device.

Plating

  1. Warm LB agar plates containing chloramphenicol (for pGGAselect) at 37°C for 15 min.
  2. Mix the cells thoroughly by flicking the tube and inverting, then spread 100 µl outgrowth onto each plate.
  3. Incubate the plates overnight at 37°C, or 24 hrs at 30°C, or 48 hrs at 25°C.

Figure 1: One-pot Golden Gate assembly of 52 fragments into a destination vector


(A) Schematic of the 52 fragment lac operon cassette assembly. Assembly inserts were generated by PCR amplification and assembled into a destination vector containing an antibiotic resistance marker. (B) Example outgrowth plate used for colorimetric scoring by reverse blue-white screening. Correctly assembled 52 insert constructs form blue colonies upon cellular transformation and incorrectly assembled constructs produce white colonies. (C) Results of the assembly reactions. Three replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per 100 μL of E. coli outgrowth plated (0.2 μL of the assembly reaction). On average, 49% of the observed transformants harbored correctly assembled constructs. Pryor, J. M. et al. (2020) bioRxiv https://doi.org/10.1101/2020.12.22.424019


 

References