2012 System for gene insertion
Researchers in the area of microbiology at the University of the Balearic Islands have developed and patented a system for gene transfer by mini transposition and its use for stable gene insertion of foreign DNA into the genome of bacteria. Employing this system it will be possible to introduce new genes and create new functions of the selected host.
This process uses the in trans action of ISPpu12, a strongly regulated IS after conjugative interaction stimulus, to insert genetic material of interest in the host bacteria. Taking advantage of such activation only during conjugation, IR-DNA fragment-IR structures of up to 10-kb can be easily inserted in a stable way in the chromosome of the bacteria permitting two or three consecutive processes.
Genetic recombination by transposing DNA fragments can be an effective tool to achieve desired characteristics in an organism.
- This process permits host bacteria achieve almost any metabolic process or phenotype which is codified in a known DNA fragment. It is a very fast, easy and reliable way for inserting large genetic fragments of interest (up to 10 kb) in a host bacterium
- The system can work without antibiotic reporter genes to screen for transference of genetic material. Therefore, antibiotic pressure is not needed for selection which allows a considerable reduction in costs when applied to industrial quantities and reduces the risk of spreading antibiotic resistance genes
- Frequencies of genetic acquisition are really high (between 10-4-10-6, depending on the size of genetic insert), avoiding alternative and non-desirable processes, and saving screening time
- The genetic material acquired is integrated in the chromosome of the bacteria. This means that they are stable insertions, avoiding easy losses like happens when inserted in plasmid structures.
- Insertion is produced mainly in one copy (although more copies can be found), avoiding multicopy insertions produced when plasmids are used (up to 20-50 copies). Such high number of copies can involve toxicity and decrease hosts fitness and growth rates.
- In comparison with other methods, its novelty resides in the mechanism for integration of the genetic material in the host, avoiding unstable plasmids or low-frequency recombination processes. As DNA integration usually proceed in monocopy, plausible toxicity effects produced by foreing DNA or multicopy DNA integration are avoided
- Integration frequences obtained with our method are, at least in our model strain (P. stutzeri AN10 harbouring ISPpu12), 3-fold higher than the ones obtained for similar trans-acting transposase procedures (i.e., miniTn5 transposons harbouring antibiotic resistance genes)
- By conjugation, IR-DNA-IR structures of up to 10-kb can be easily inserted in a stable way in the chromosome of the bacteria. As this process can be repeated several times, it big amounts of foreings DNA can be introduced in the host and, therefore, host metabolic capabilities can be easily enhanced
The process has already been well characterized, and the tools necessary for applying it have been synthesized and tested. The method is protected by a patent and is available for license.
The applications of the process we propose are as diverse as the needs for innovating bacterial strains to face an increase of their fitness or convert bacteria in a biotechnological tool to catabolize or synthesize any kind of substrate.
As an example of the potential of the process we have tested both possibilities achieving:
- An increase in growth capability at low temperatures (4ºC)
- Introduce the necessary genes in this strain to confer it the ability of using a contaminant (alkanes, linear hydrocarbons)