35-Fragment Golden Gate Assembly using BsmBI-v2 (NEB #E1602)


Overview

Golden Gate Assembly of 35 fragments using BsmBI-v2 restriction enzyme with T4 DNA Ligase and subsequent transformation into NEB 10-beta Competent E. coli (High Efficiency). 

Materials

  • NEB Golden Gate Assembly Kit (BsmBI-v2) (NEB #E1602)
  • 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 (BsmBI-v2) (NEB #E1602)

Reaction set-up

Set up assembly reactions as follows:

REAGENT ASSEMBLY REACTION
pGGAselect Destination Plasmid(1)*, 75 ng/μl 1 μl
Amplicon Inserts(2) 3 nM(3) each amplicon (final concentration)
T4 DNA Ligase Buffer (NEB #B0202) (10X) 2 μl
NEB Golden Gate Enzyme Kit (BsmBI-v2) (NEB #E1602) 2 μl 
Nuclease-free H2O (NEB #B1500) to 20 μl(4)

* or user provided


(1) Destination vector must possess BsmBI 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 BsmBI 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) Can be increased to 25 μl volume if required due to DNA component volumes; add additional 0.5 μl T4 DNA Ligase Buffer
(10X).

Reaction assembly protocol
SUGGESTED ASSEMBLY PROTOCOL
(42°C, 5 min → 16°C, 5 min) x 30(5) → 60°C, 5 min → 4°C(6)

(5) Additional cycles 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: High capacity Golden Gate assembly with T4 DNA ligase and BsmBI-v2.



(A) Schematic of the 35-fragment lac operon cassette test system (B) Results of the assembly reactions. Four replicate experiments were carried out to quantify the number of colony-forming units harboring correct and incorrect assembly products per 50 μL of E. coli outgrowth plated (0.1 μL of the assembly reaction). On average, 71% of the observed transformants harbored correctly assembled products. (C) Representative agar plate with blue and white colonies. Blue transformants harbor correct assembly constructs, and white transformants harbor inaccurate assembly products. Pryor, J. M. et al. (2020) PLoS Onehttps://doi.org/10.1371/journal.pone.0238592


 

References