Design your own bacteria in one step

The production of designer bacteria for biotechnology processes used to be an inefficient one. Now, University of Adelaide researchers have developed a new method that is simple and fast.

Led by Dr Keith Shearwin of the university’s School of Molecular and Biomedical Science, in collaboration with Stanford University, the researchers have developed a one-step bacterial genetic engineering process called ‘clonetegration’, described in the journal ACS Synthetic Biology. Their research facilitates faster development of designer bacteria used in therapeutic drug development, such as insulin, and other biotechnology products.

Designer bacteria are produced by integrating extra pieces of genetic material into the DNA of bacteria - eg, E. coli, which is often used in biotechnology - so that the bacteria will make a desired product.

“New genes or even the genetic material for whole metabolic pathways are inserted into the bacteria’s chromosome so that they produce compounds or proteins not normally produced. Insulin is an example of a therapeutic product produced in this way,” said Dr Shearwin.

Dr Shearwin explained that the new process is based on the integration in single copy of a DNA sequence of interest into a defined position within the bacterial chromosome.

“The process takes advantage of bacteriophage attachment sites which are present throughout bacterial genomes and uses the corresponding phage integrase enzyme to catalyse the site-specific integration reaction,” he said.

While the existing process works on the same principle, Dr Shearwin says it uses two different plasmids and a number of steps to perform the integration, taking several days. The remodelled plasmid system “allows a one-step process where a cloning reaction or DNA assembly reaction can be transformed directly into the bacterial strain”, so it can be completed overnight.

As well as speeding up the process, clonetegration enables multiple rounds of genetic engineering on the same bacteria and simultaneous integration of multiple genes at different specific locations.

“This will become a valuable technique for facilitating genetic engineering with sequences that are difficult to clone, as well as enable the rapid construction of synthetic biological systems,” Dr Shearwin said.

Dr Shearwin said the system should work in most types of bacteria “since almost all bacteria are susceptible to bacteriophage infection and have the requisite attachment site sequences within their genomes”. Apart from E. coli, it has also been tested in Salmonella typhimurium, where it works well.

Furthermore, he noted, “any gene or series of genes can be inserted into the bacterial chromosome by our method”, including those genes or larger pathways which can’t be maintained within bacterial cells on multicopy plasmids because of toxicity problems.

The molecular tools needed for the clonetegration process are being submitted to Addgene, a DNA repository, and the system should be freely available midyear for ongoing research and development.