Our laboratory is at the forefront of lactic acid bacteria genome editing. Since several projects in our laboratory focus on the gut symbiont Limosilactobacillus reuteri, we use this species as our model to develop new genome editing technologies.
Recombineering. We routinely apply single-stranded DNA recombineering (SSDR) to modify the bacterial chromosome. A short oligonucleotide is introduced in bacterial cells that temporarily express a recombinase protein. The recombinase helps to stabilize, and possibly protect, the oligonucleotide. The oligonucleotide serves as an Okazaki fragment, and is extended by DNA polymerase into a new strand. User-defined non-homologous bases in the oligonucleotide will result in targeted base changes in the chromosome.
CRISPR. In addition, we pioneered the application of CRISPR-Cas-assisted recombineering in lactic acid bacteria. CRISPR-Cas serves as a tool to enrich the recombinant genotype in a population. This opens up the exciting opportunity to identify low-efficiency mutagenesis events. For example, implementing CRISPR-Cas means we can identify up to 1kb chromosomal deletions generated by a single oligonucleotide. We also demonstrated we can modify a single codon to encode 19 additional amino acids. All mutants were generated by a single person within a couple of days.
Counterselection system. While SSDR and CRISPR-Cas are very powerful technologies, the efficacy by which these tools function vary per strain. Therefore, we developed a counterselection system that functions in many members of the family Lactobacillaceae. Unlike most counterselection systems, our platform does not require prior genome editing and can be combined with a liquid-based approach to generate insertions or deletions in a matter of days.
For more in-depth information on these genome editing technologies, we refer to these select publications.
Finally, we took the plunge! The van Pijkeren laboratory is on Twitter. We hope to connect with many colleagues in the field, share interesting articles and tweets, and -of course- share our latest news and …May 25, 2017
The Van Pijkeren Laboratory is featured in several news articles that were recently published. MIT Technology Review published the article Edible CRISPR could replace antibiotics. Around the same time, the article Antibiotic alternatives, delivered by friendly microbes appeared …April 21, 2017
Looking back at 2016, it has been an exciting year, no doubt! For starters, we had our website launched to showcase our work! We are very proud of the final product, and we will continue …December 29, 2016
We’re very excited to have a new website! Check it out to meet the members of the van Pijkeren Laboratory, and to learn more about our research. We hope to keep you up to …September 13, 2016