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Related Experiment Video

Updated: Mar 25, 2026

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

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A BioBrick™-Compatible Vector for Allelic Replacement Using the XylE Gene as Selection Marker.

Michela Casanova¹, Lorenzo Pasotti¹, Susanna Zucca¹

¹Department of Electrical, Computer and Biomedical Engineering, Laboratory of Bioinformatics, Mathematical Modelling and Synthetic Biology, University of Pavia, 27100 Pavia, Italy ; Centre for Health Technologies, University of Pavia, 27100 Pavia, Italy.

Biological Procedures Online

|February 16, 2016

Summary

We developed pBBknock, a novel BioBrick™-compatible vector for marker-less allelic replacement in Escherichia coli. This versatile tool simplifies genome engineering for researchers and iGEM teams, offering broader applicability than existing methods.

Keywords:

Allelic replacement BioBrick Knockout Standard vector XylE

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Area of Science:

Synthetic Biology
Molecular Biology
Microbial Engineering

Background:

Marker-less allelic replacement in Escherichia coli commonly uses circular plasmid-mediated homologous recombination.
Existing methods face limitations such as high false positive rates and restricted strain or media applicability.
BioBrick™-compatible solutions for homologous recombination are currently unavailable.

Purpose of the Study:

To develop a novel BioBrick™-compatible vector for marker-less allelic replacement in Escherichia coli.
To overcome limitations of existing homologous recombination methods.
To provide a versatile tool for genome engineering in various E. coli strains and media.

Main Methods:

Introduction of pBBknock, a novel BioBrick™-compatible vector.
Utilizing a temperature-sensitive replication origin for marker-less genome engineering.
Employing two homologous recombination events with positive selection (chloramphenicol resistance) and a colorimetric screen (xylE gene).

Main Results:

Demonstration of pBBknock for marker-less allelic replacement in Escherichia coli.
Successful deletion of the lactate dehydrogenase gene in E. coli W.
pBBknock enables efficient screening of desired genomic modifications.

Conclusions:

pBBknock offers a broader application range compared to existing plasmid-based solutions.
The vector is not limited to specific E. coli strains or growth media.
pBBknock is a versatile solution for researchers and iGEM competition teams utilizing BioBrick™ assembly.