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Determining the Role of the luxR homolog in Mycobacterium avium subsp. paratuberculosis in Bacterial Invasion of Bovine Epithelial Cells. Presented by: Lauren Shin Mentor: DR. Luiz Bermudez Microbiology DEPARTMENT. - PowerPoint PPT Presentation
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PRESENTED BY: LAUREN SHINMENTOR: DR. LUIZ BERMUDEZMICROBIOLOGY DEPARTMENT
Determining the Role of the luxR homolog in Mycobacterium avium subsp. paratuberculosis in Bacterial Invasion of Bovine Epithelial Cells
http://microbewiki.kenyon.edu/index.php/File:EM_Scan_Paratuberculosis.jpg
Mycobacterium avium subsp. paratuberculosis (MAP) and Johne’s Disease
MAP Agent of Johne’s disease in cattle and other ruminants
Infects and grows within lining of intestine
Passed through the milk of infected animals
Mortality rate = 100%No treatment or
efficient vaccine http://www.johnes.org/dairy/_Holstein_front.html
Significance of Our Research
Provides useful information for characterizing and determining more desirable vaccine targets for Johne’s disease
2007 study by the USDA estimated that Johne’s disease has an approximately $200 million/year economical impact on beef and dairy industry
Further research on how LuxR contributes to invasion in the early stages of the disease
Background Research
MAP can be delivered to host by milkMAP exposed to milk greater efficiency of
invasionluxR homolog gene also significantly up-
regulated when exposed to milkLuxR regulates transcription of many other
genesThese gene homologues alter bacterial cell
wall composition may assist in invasion
Our Research Project
Aim: To determine whether or not the luxR homolog gene plays a direct role in invasion of MAP into epithelial cells
Approach: Overexpress LuxR and its dependent genes in normally non-invasive Mycobacterium smegmatis and observe its effect on invasion.
Our Research Project
Hypothesis: The luxR homolog gene and its dependent genes in MAP play a direct role in the invasion of MAP into epithelial cells.
Prediction: If LuxR and its dependent genes are overexpressed in M. smegmatis, the mycobacterium will invade epithelial cells with greater efficiency than a wild-type invasion.
Our Genes
Cloning three genes: MAP0482, MAP0483, and MAP4088
MAP0482 and MAP0483 make up luxR homolog in M. avium subsp. avium
LuxR regulates the transcription of MAP4088 and MAP1203, both hypothetical invasion proteins
Methods
Step 1: Clone luxR related genes into pLDG13A 5-step process
1. PCR
2. Digestion
3. Ligation
4. Transformation
5. Screening/
Sequencing
Designing Our Primers and PCR
Forward Primer:
Reverse Primer:
Restriction Site (HindIII)
Ribosomal Binding
Site
HIS-Tag
Forward Sequence
Reverse Sequence
Restriction Site (KpnI)
http://www.mun.ca/biology/scarr/PCR_simplified.html
Cloning
Digest with restriction enzymesLigate with T4 DNA ligaseTransformation by electroporation into E. coliPlate on Kanamycin platesScreen by colony PCR or digestion to
visualize clonesVerify sequence
Methods
Step 2: Transform plasmid with inserted
gene into M. smegmatis by
electroporation Step 3: Protein Gel and Western Blotting to
verify expression of genes in M. smegmatis
Methods
Step 4: Perform invasion assays in which transformed M. smegmatis is allowed to infect epithelial cells
http://www.sz-wholesaler.com/p/893/905-1/24-well-cell-culture-plate-406360.html
Invasion Assay Protocol
Add bacteria to epithelial
cells
Incubate (1h, 3h) in 37°C to allow invasion
Add antibiotics
and wash off extracellular
bacteria
Lyse cells with detergent to
release bacteria
Serial dilute lysates and
plate
Difficulties with Cloning…
PCR Amplification?
Problem: No amplification of genes
Possible Explanation: HIS-tag primers contain unspecific sequences; cannot anneal with such a large template of genomic DNA
4088 0482 0483Ladder
PCR Amplification?
Proposed Solution: 2-step PCR amplification
Outcome: Still faint or inconsistent bands
Gene gDNA
His-tag
Forward Primer
Reverse Primer
Gene Gene
PCR Amplification?
Possible Explanation #2: Genes 4088, 0482, and 0483 are GC rich; difficult to PCR because they can form secondary structures like hairpins and have higher melting temperatures
Proposed Solution: Use GC-RICH PCR System Contains DMSO, Polymerase from GC-rich organism
Outcome: Genes amplified!
_____0483______L
Continued Cloning
Problem: After digesting and ligating the amplified genes, the screens showed empty vectors with no insert.
Possible Explanations: 1. The restriction enzymes may not be cutting
completely, leaving uncut plasmid2. The plasmid may be re-ligating together
Still Troubleshooting
Proposed Solutions:1. Shrimp Alkaline Phosphatase (SAP)
dephosphorylate plasmid to prevent self ligation
2. Include controls for ligations; without insert3. Make sure digestion and ligation is working
correctly with pLDG13
Another Important Gene
MAP1203 LuxR regulated geneAlready cloned into pLDG13Attempting to verify expressionContinued with invasion assay
Untransformed M. smegmatis M. smegmatis transformed with empty vector,
PLDG13 M. smegmatis transformed with wild type 1203 clone M. smegmatis transformed with ΔRGD 1203 mutant
Invasion Assay Results
Smeg PLDG13 WT 1203 ∆RGD0
0.1
0.2
0.3
0.4
0.5
0.6
Percent Invasion of M. smegmatis in MDBK Cells at 1 hour and at 3 hours
1h3h
% I
nvas
ion
Future Direction
Continue troubleshooting to obtain clones
Verify expression of genes
Invasion Assay
Binding Assay
Yeast Two-Hybrid System Identify receptor protein to which the bacterium binds
Acknowledgements
Dr. Luiz BermudezDr. Kevin AhernJamie EvermanBermudez Lab
Howard Hughes Medical InstituteUniversity Honors College
Cripps