Mycobacterial species are diverse organisms, classified into tuberculous and nontuberculous mycobacteria (NTM). Mycobacterium tuberculosis has been thoroughly investigated, but pathogenic NTM have not. The identified technology gap for studying potential antibiotic targets across mycobacteria is that there is not a tool developed for efficiently creating bacterial clones containing these genes with a reporter system to evaluate CRISPR interference (CRi) knockdowns. CRi is a quick and simple way to silence genes. In this study, Golden Gate (GG) cloning compatible Lux reporter plasmids were engineered for the efficient cloning of target genes as transcriptional fusions with luxAB, a luminescent reporter, for use with CRi. Additionally, a CRi plasmid was designed with a Giles integration site so that it could be integrated into the mycobacterial genome with the reporter plasmid, but at a different location. Based on current research, it seems that mycobacterial L,D-transpeptidase enzymes (Ldts), involved in peptidoglycan synthesis, are potential targets for the drug class known as β-lactams and should be further explored. Ldt 2 is of particular interest as research indicates that it may be involved in pathogenicity; therefore, GG cloning of M. smegmatis (Msm) Ldt 2 was performed using the designed GG plasmid. Constructing the GG plasmid (pMV306hsp+luxG13, GG pMV) as well as the CRi + Giles integration plasmid (pLJR962 + pML1357, CRi + Giles) was successful; however, the evaluation of the luminescent reporter with CRi knockdown has yet to be performed.

Thesis Completion




Thesis Chair/Advisor

Rohde, Kyle H.


Bachelor of Science (B.S.)


College of Medicine


Burnett School of Biomedical Sciences

Degree Program

Biomedical Sciences



Access Status

Campus Access

Length of Campus-only Access

5 years

Release Date