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Genetics / Genomics JoVE (Open Access) Citable · DOI

Creation of a Dense Transposon Insertion Library Using Bacterial Conjugation in Enterobacterial Strains Such As Escherichia Coli or Shigella flexneri

DOI: 10.3791/56216-v
What you'll learn
  • ✓ Perform bacterial conjugation to transfer transposons into enterobacterial genomes
  • ✓ Create a high-density transposon insertion library with hundreds of thousands of unique mutants
  • ✓ Pool and isolate individual bacterial mutants from conjugation library
Protocol

Presented here is a simple method for creating a high-density transposon insertion library in Escherichia coli or Shigella flexneri using bacterial conjugation. This protocol allows the creation of a collection of hundreds of thousands of unique mutants in bacteria by the random genomic insertion of a transposon.

Difficulty
intermediate
Total time
~3–5 days (including mating, selection, and pooling)
Model organism
Escherichia coli, Shigella flexneri
Biosafety
BSL-1

Steps

1
Set up bacterial conjugation mating pair

Prepare donor and recipient bacterial strains for conjugation. Combine cultures to enable horizontal transfer of the transposon from donor to recipient cells.

▶ 00:44
2
Overview of transposon library creation method

Understand the overall experimental strategy for generating random genomic transposon insertions and the resulting mutant library composition.

▶ 05:38
3
Pool conjugation products into library

Combine and consolidate all transposon-inserted mutant colonies from the conjugation into a single pooled library for downstream use.

▶ 08:52
4
Analyze and interpret library results

Examine the characteristics of the generated transposon insertion library, including insertion density and mutant diversity.

▶ 10:25
5
Apply transposon library for downstream studies

Use the dense insertion library to screen for genes involved in specific phenotypes or survival conditions in enterobacterial strains.

▶ 10:53
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