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

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

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

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

Plasmids in this study.

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