Home › Biochemistry › Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly
Biochemistry JoVE (Open Access) Citable · DOI

Generation of Plasmid Vectors Expressing FLAG-tagged Proteins Under the Regulation of Human Elongation Factor-1α Promoter Using Gibson Assembly

DOI: 10.3791/52235-v
What you'll learn
  • ✓ Design and construct custom plasmids using Gibson assembly methodology
  • ✓ Generate FLAG-tagged protein expression vectors for mammalian cells
  • ✓ Apply in silico design, PCR amplification, and molecular cloning techniques
  • ✓ Verify successful cloning through transformation and colony screening
Protocol

Synthesis of custom plasmids is labor and time consuming. This protocol describes the use of Gibson assembly cloning to reduce the work and duration of custom DNA cloning procedure. The protocol described also produces reliable tagged protein constructs for mammalian expression at similar cost to the traditional cut-and-paste DNA cloning.

Difficulty
intermediate
Total time
~3-4 days (including overnight bacterial culture and colony verification)
Biosafety
BSL-1

Steps

1
Design final plasmid and overlapping primers in silico

Plan the target plasmid architecture and design primers with overlapping sequences for Gibson assembly. This computational step precedes all wet-lab work.

▶ 01:30
2
Amplify DNA fragments via PCR

Use designed primers to amplify the plasmid backbone and insert fragments from template DNA. PCR generates the overlapping DNA segments required for Gibson assembly.

▶ 03:58
3
Purify amplified DNA fragments using magnetic beads

Clean PCR products with magnetic bead-based purification to remove polymerases, primers, and other contaminants. Provides DNA suitable for assembly cloning.

▶ 05:44
4
Perform Gibson assembly and transform into E. coli

Combine purified DNA fragments in a Gibson assembly master mix containing exonuclease, polymerase, and ligase. Transform the assembled plasmid into competent E. coli and select transformants on antibiotic plates.

▶ 06:58
5
Verify successful cloning of tagged protein constructs

Analyze results by colony screening, plasmid miniprep, and restriction digest/sequencing to confirm correct assembly of FLAG-tagged CstF-54 and CstF-64 variants under EF1α promoter control.

▶ 08:21
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