Contribution to the study of the conjugational mobilization provoked from the natural plasmid PZA1003 of Zymomonas mobilis nciμb 11163 (Master thesis)
Zymomonas mobilis is a bacterium able to grow under both aerobic and anaerobic conditions. It is of great biotechnological interest due to the higher yield of ethanol production in comparison with the fungus Saccharomyces cerevisiae. This high yield of ethanol production is accompanied by less biomass production and the lack of need for oxygen for the growth of its cells. A strategy used for the genetic improvement of this bacterium is the creation of cloning vectors that bear regions of its natural plasmids, since it is known that its strains contain many natural plasmids with a variety of sizes. In the present study, the ability of the natural plasmid pZA1003 of Z. mobilis NCIMB 11163 to mobilize has been examined. More precisely, a new cloning vector has been constructed, based on the broad host range plasmid pBR328, in which regions of the plasmid pZA1003 have been added. These regions are essential for its replication in its host and for its mobilization trough helped conjugation, the same way it happens between Escherichia coli cells. The success of the construction of this new cloning vector, named pBRZArm, has been validated through Southern hybridization and Polymerase Chain Reaction. The ability of pBRZArm to mobilize has been established primarily with helped conjugation between Eschericia coli cells. Consequently, it has been used for conjugation with Z. mobilis CP4Rif cells as recipients, with the aid of the helper plasmid pRK2013, which belongs to the INCP incompatibility group. The success of this second conjugation has been confirmed through Southern hybridization and transformation of E.coli cells with the conjugated CP4Rif cells. pBRZArm has been successfully transferred through conjugation, as well as with a great frequency. Also, it is almost 100 % stable in Z. mobilis cells for at least 130 generations, under non- selective conditions. According to those qualities, pBRZArm is a cloning vector appropriate for expression of foreign genetic information in Z. mobilis. Lastly, the ability of pBRZArm to mobilize between Z. mobilis cells has been examined, more precisely between CP4Rif and a tetracycline resistant derivative of ATCC 10988. Although colonies of the recipient cells have been observed, that were apparently resistant to the antibiotic of selection (chloramphenicol), plasmid DNA analysis and transformation of E. coli cells showed no presence of pBRZArm. This indicates that the mobilization abilities of pZA1003 cannot be induced in the presence of Z. mobilis CP4Rif natural plasmids, either due to incompatibility between the two, or to the lack of conjugational function in them. However, conjugative ability of endogenous plasmids of other Z. mobilis strains is yet to be examined.
|Institution and School/Department of submitter:||Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Βιολογικών Εφαρμογών και Τεχνολογιών|
|Subject classification:||Zymomonas mobilis|
|Appears in Collections:||Διατριβές Μεταπτυχιακής Έρευνας (Masters)|
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|Μ.Ε. ΜΑΥΡΟΜΜΑΤΗ ΜΑΡΙΑ 2018.pdf||3.81 MB||Adobe PDF||View/Open|
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