Inter-annual variations of planktonic food webs in the northern Adriatic Sea.

13nonenoneFONDA S.; MILANI L; BORME D; DE OLAZABAL A; PARLATO S; PRECALI R; KRAUS R; LUCIC D; NJIRE J; TOTTI C; ROMAGNOLI T; POMPEI M; CANGINI MFonda, Serena; Milani, L; Borme, D; DE OLAZABAL, A; Parlato, S; Precali, R; Kraus, R; Lucic, D; Njire, J; Totti, C; Romagnoli, T; Pompei, M; Cangini, M

Engineering More Stable, Selectable Marker-Free Autoluminescent Mycobacteria by One Step

<div><p>In our previous study, we demonstrated that the use of the autoluminescent <i>Mycobacterium tuberculosis</i> as a reporter strain had the potential to drastically reduce the time, effort, animals and costs consumed in evaluation of the activities of drugs and vaccines in live mice. However, the strains were relatively unstable and lost reporter with time without selection. The kanamycin selection marker used wasn’t the best choice as it provides resistance to amino glycosides which are an important class of second line drugs used in tuberculosis treatment. In addition, the marker could limit utility of the strains for screening of new potential drugs or evaluating drug combinations for tuberculosis treatment. Limited selection marker genes for mycobacterial genetic manipulation is a major drawback for such a marker-containing strain in many research fields. Therefore, selectable marker-free, more stable autoluminescent mycobacteria are highly needed. After trying several strategies, we created such mycobacterial strains successfully by using an integrative vector and removing both the resistance maker and integrase genes by Xer site-specific recombination in one step. The corresponding plasmid vectors developed in this study could be very convenient in constructing other selectable marker-free, more stable reporter mycobacteria with diverse applications.</p></div

Overview: the 15th Jkuat Scientific, Technological and Industrialization Conference

The conference provided a forum through which the university showcases ongoing contributions it is making to the society; created a forum for constantly improving the University’s approach to development-oriented scientific research, as it strives to remain a leader in this area; provided a forum for research peers from local and international institutions to discuss, share and publish vital information; provided an opportunity for the industrial/business sectors and policymakers to interact with researchers, so as to get new ideas and products for infusion into the production system and research and provoked policy makers to appreciate the need for substantial and long-term investments in scientific research, innovation and industrialization

The plasmids constructed in this study for transforming into mycobacteria to create unmarked autoluminescent mycobacteria.

<p><i>oriE</i>, origin region of <i>E</i>. <i>coli</i>; <i>Hsp60</i>, the strong mycobacterial promoter; <i>luxCDABE</i>, the operon for producing autoluminescence; <i>bla</i>, ampicillin resistance gene; <i>Kan</i>, KAN resistance gene; <i>res</i>, the transposonγδ resolvase action site; <i>attP</i>, mycobacteriophage L5 attachment site; <i>int</i>, integrase gene; <i>int’</i>, the remaining part of integrase gene; <i>attB</i>, attachment site from the mycobacterial genome corresponding to <i>attP</i>; <i>oriM</i>, origin region of mycobacteria; <i>Hyg</i>, HYG resistance gene; <i>dif</i>, the recombinases XerCD action site.</p

Plasmids in this study.

<p>AMP: ampicillin; KAN: kanamycin; HYG: hygromycin.</p><p>Plasmids in this study.</p

Photograph of the more stable, selectable marker-free, autoluminescent BCG (UABCG).

<p>Photograph of the more stable, selectable marker-free, autoluminescent BCG (UABCG).</p