Creation of a Reporter Gene Fusion Vector for the Localism of Mycobacteria Proteins
Location
CSU 253/4/5
Start Date
4-4-2011 1:30 PM
End Date
4-4-2011 3:00 PM
Student's Major
Biological Sciences
Student's College
Science, Engineering and Technology
Mentor's Name
Timothy Secott
Mentor's Department
Biological Sciences
Mentor's College
Science, Engineering and Technology
Description
Transmission of Mycobacterium avium subsp. paratuberculosis (Mpt) within cattle has become an economic issue for dairy farmers. Mpt is the causative agent of Johne‘s disease, a chronic, fatal intestinal disease of ruminants. Intracellular multiplication of Mpt causes the release of virulence factors and enteritis, inevitably killing the host. As Mpt grows it is excreted, subsequently infecting other livestock. Infected cattle fail to show symptoms initially and often go undiagnosed. Since these cattle are not removed from the herd immediately it allows for the uncontrolled spread of disease. Understanding how Mpt responds to its environment may lead to more efficient methods of disease control. Our primary goal was to construct a plasmid that would allow determination of when, where, and under what conditions various proteins concentrate in Mpt. Plasmid pMV261, a mycobacterial shuttle vector was cut with enzymes HindIII and HpaI. A plasmid, pEGFP-N1 containing a coding region for enhanced green fluorescent protein (eGFP) was cut with the enzymes HindIII and NsiI to release a 1709bp fragment of DNA containing the entire coding sequence of eGFP and multiple restriction sites. A ligation reaction was conducted to fuse the eGFP coding region into pMV261. A total of 6 new restriction sites were added to the plasmid pMV261 as a result of this addition. This newly constructed plasmid was inserted into Mycobacterium smegmatis for analysis and determining the success of transformation. Subsequent findings from transformations can provide a greater understanding of Mpt and the proteins employed when responding to stressors within the host.
Creation of a Reporter Gene Fusion Vector for the Localism of Mycobacteria Proteins
CSU 253/4/5
Transmission of Mycobacterium avium subsp. paratuberculosis (Mpt) within cattle has become an economic issue for dairy farmers. Mpt is the causative agent of Johne‘s disease, a chronic, fatal intestinal disease of ruminants. Intracellular multiplication of Mpt causes the release of virulence factors and enteritis, inevitably killing the host. As Mpt grows it is excreted, subsequently infecting other livestock. Infected cattle fail to show symptoms initially and often go undiagnosed. Since these cattle are not removed from the herd immediately it allows for the uncontrolled spread of disease. Understanding how Mpt responds to its environment may lead to more efficient methods of disease control. Our primary goal was to construct a plasmid that would allow determination of when, where, and under what conditions various proteins concentrate in Mpt. Plasmid pMV261, a mycobacterial shuttle vector was cut with enzymes HindIII and HpaI. A plasmid, pEGFP-N1 containing a coding region for enhanced green fluorescent protein (eGFP) was cut with the enzymes HindIII and NsiI to release a 1709bp fragment of DNA containing the entire coding sequence of eGFP and multiple restriction sites. A ligation reaction was conducted to fuse the eGFP coding region into pMV261. A total of 6 new restriction sites were added to the plasmid pMV261 as a result of this addition. This newly constructed plasmid was inserted into Mycobacterium smegmatis for analysis and determining the success of transformation. Subsequent findings from transformations can provide a greater understanding of Mpt and the proteins employed when responding to stressors within the host.
Recommended Citation
Edwinson, Adam. "Creation of a Reporter Gene Fusion Vector for the Localism of Mycobacteria Proteins." Undergraduate Research Symposium, Mankato, MN, April 4, 2011.
https://cornerstone.lib.mnsu.edu/urs/2011/poster-session-C/5
