University of Tampere, BioMediTech
Tuberculosis is a bacterial infection that is caused by Mycobacterium tuberculosis and is often found in human lungs. For this research, it is more feasible to use Mycobacterium marinum, a zebrafish pathogen that is less contagious than M. tuberculosis. With this experiment we want to optimize the production of knockout strains of Mycobacterium marinum and the identification of true knockout strains among the colonies. Electroporation of a plasmid that is unable to replicate in mycobacteria is used to construct possible knockout strains of the mycobacteria via homologous recombination consisting of two subsequent cross-over events. As a result, the gene-of-interest will be replaced by a hygromycin resistance gene. The plasmid also contains other marker genes (LacZ and sucrose sensitivity), these genes can be used for colony PCR and sucrose test of potential single- and double-crossover strains. During the optimisation of the PCR protocol, we observed small facts to look at: the use of the correct positive control (pTEC27 versus maxiprep plasmid), time of the growth of the bacteria and the “age” of the culture or the colonies on the plate of the mycobacteria. Secondly, the sucrose test is a suitable way to test for the sensitivity of single crossover bacteria for sucrose. In addition to the optimization, possible knockouts were created (MMAR_0514 and MMAR_4524). The obtained results of the marker gene tests are looking very promising. Since the results of the sucrose test, we know that the chance is bigger that the strains (MMAR_0514 and MMAR_4524) are a single-crossover. Furthermore, the possible knockout strains to confirm the insertion site of the hygromycin resistance gene, in the bacteria. After this, the created knockouts could be tested for infectivity and ability to reactivate by injecting them into the zebrafish.
Address
Biokatu 6
Tampere, Finland
+358 40 190 1664 Finland |
Biokatu 6
Tampere, Finland
+358 40 190 1664 Finland |
Contacts
Traineeship supervisor
Mika Rämet
|
Traineeship supervisor
Laura Vesala
|
Traineeship supervisor
Matti Nykter
|
Traineeship supervisor
Henna Myllymäki
|
Meri Uusi-Mäkelä
|