Faculty of Food Technology and Biotechnology, University of Zagreb
Abstract
Flippase expression was carried out in Zymomonas mobilis strain ZM4. The FRT-flanked selection marker gene was first integrated into the ZM4 chromosome by homologous recombination. The Saccharomyces cerevisiae flp gene was then introduced under the control of the ZM4 gap gene promoter (Pgap, encoding glyceraldehyde-3-phosphate dehydrogenase) or the λ bacteriophage cI857-pR contained in the broad-host-range cloning vector pBBR1-MCS-2. This study demonstrated that flp was expressed and that the deletion frequency of the FRT-flanked marker gene was very high (approx. 100 %). In addition, the flp gene expression vector could be conveniently removed from the resulting unmarked Z. mobilis mutants by serially transferring the cells three times into antibiotic-free medium, thereby establishing an efficient method for constructing unmarked Z. mobilis mutants.U ovom je radu u soju bakterije bakterije Zymomonas mobilis ZM4 eksprimirana flipaza iz kvasca Saccharomyces cerevisiae. Najprije je homolognom rekombinacijom u bakterijski kromosom ugrađen selektivni biljeg omeđen FRT sekvencijama. Potom je u bakteriju unesen plazmid pBBR1MCS-2 koji sadrži kvaščev gen Flp pod regulacijom promotora gena gap iz soja ZM4 (Pgap, koji kodira za gliceraldehid-3-fosfat dehidrogenazu) ili cI857-PR iz bakteriofaga λ. Gen Flp uspješno je eksprimiran, te je učestalost gubitka selektivnog markera omeđenog FRT sekvencijama iznosila približno 100 %. Osim toga, vektor za ekspresiju gena Flp lako je uklonjen trostrukim precjepljivanjem na podlogu bez antibiotika, pa se može zaključiti da je razvijena učinkovita metoda za uklanjanje selektivnog biljega iz transformanata bakterije Zymomonas mobilis
