Justification:
 
Current methods involve inefficient intravenous procedures, and unnecessary risk to veterinarians administering the vaccine. There have been steps within the research community to advance an alternative vaccine using a non-pathogenic microbe that expresses the B. abortus surface antigens (citation/s). This probiotic vaccine would activate immunity in the rumen of the gut allowing safe protection from B. abortus.  The live vaccine in the form of Lactococcos lactis is a possible route to immunity without the risk of fetal abortion or human infection. From research we are able to gain knowledge of some conditions that will be faced, however there still lies many unknowns. Initially we will be seeking to better understand the growth curve of the transformed L. lactis [RW1] in comparison to the wild type strain. Data on the growth of L. lactis can be used to compare the different rates of growth within the rumen versus normal conditions, and highlight the metabolic strain the probiotic will face. These methods simulate rumen conditions, and the pressures that the probiotic will experience during its antigen expression.
Objectives:
• Test the viability of live Lactococcus lactis [RW2] in the rumen as a vector for Brucellosis vaccination.
• Monitor the environmental and generational stability of the expression plasmid in Lactococcus lactis.
• Understand the effects the rumen will have on the nisin inducible expression system being used in Lactococcus lactis.
• Evaluate the possibilities of Gram-positive to Gram-negative gene transfer when using a plasmid containing antibiotic resistance.
 
 
Hypothesis:
a) We hypothesize that there will be persistent stability of the Nisin-Inducible Expression system (NICE) vector plasmid inside the Lactococcus lactis and the species itselfover changes in pH, temperature, and growth pressure, similar to what is found in rumen.
b) The stability and expression of the NICE plasmid will not be reduced over generations. 
c) Growing common enteric bacteria and pathogens alongside the vector Lactococcus lactis will not result in DNA exchange involving the NICE vector. 
Specific Aims:
• Growth in normal conditions (30° Celsius, pH 6.8) will be measured using a spectrophotometer by measuring every 6 hours, plating the culture, and plotting cell count versus OD values to create growth curves.
• Growth of vector strain L. lactis will be measured in ranging temperatures of 30°-40° Celsius, pH values ranging from 5.7-7.3, and varying microbial cultures, all in M17 broth.
• Plasmid stability will be determined by observing growth on chloramphenicol M17 plates.
• Mixed cultures will be screened for gene transfer by plating M17 broth with chloramphenicol, isolating potential recipient bacteria, and testing plasmid presence via cell lysate gel electrophoresis.
Overview:  
In our experiments we will first address the stability of the plasmid, expressing the Brucella antigen, which must be proven to remain viable in the rumen. Growing the transformed strain in specific pH, temperature, and against wild type and shuttle strains will test this. The plasmid expression and viability will be tested across generations. The plasmid contains an antibiotic resistance gene, which could be absorbed by pathogens present in the bovine population. As the growth of the transformed L. lactis reaches a later stage, cell death and loss of genetic information into the extracellular fluid is a possibility. Second we will address the safety of the plasmid. We will test the possibility of transformation into other bacterial species by inserting the naked plasmid into broth cultures of the following: Salmonella enterica, Yersinia enterocolitica, Camplyobacter jejuniEscherichia coli, and Bacillus subtilis. Bacteria that acquire the plasmid vector will be selected based on growth in chloramphenicol. We will select transformed colonies to culture over generations, testing the extended stability of the NICE plasmid.[RW3] 
 Materials and Methods:   
      i.         The laboratory of G.P. Andrews of the University of Wyoming will provide bacterial stocks.
     ii.         L. lactis both the wild type and transformed strain will be grown on M17 media that is supplemented with 0.5% glucose and incubated at 30ºC(6). We will also be streaking Bacilis subtilis, E. coli from frozen stock onto TSA. From frozen stock onto blood agar plates (BPA) we will be streaking Salmonella enterica, Yersinia enterocolitica, and Campylobacter jejuni. All will be grown in their desired 37 degrees Celsius environments.   
  iii.         L. lactis will be grown on M17 media, and in M17 broth, which will both be supplemented with 0.5% glucose and incubated at 30oC.  We will utilize a pH meter and create an M17 broth and agar that is between the pH of 5.7 and 7.3. We will then grow the L. lactis wild type and transformed strain in the broth and measure culture density using a Beckman model  ?   spectophotometer. We will also use a similar procedure for testing the growth of L. lactis between the temperatures of 37.8 and 40 oC.. We will also insert the naked plasmid into broth growths of the previously streaked stomach pathogens to test for the uptake of the plasmid. We will also utilize plasmid extraction to determine the plasmids presence within the microbes (5).[RW4]       HOW MUCH “NAKED” PALSMID ARE YOU GOING TO USE AND WHAT IS THE RATIONALE FOR THE AMOUNT YOU SELECT?
iv.        Serial subcultures will be performed by inoculating 10 mL of fresh medium at a 1:100 dilution. TIME OF INCUBATION?   STATIC VS AGITATION?  TEMP?   from the previous culture. After 5 subcultures, the number of chloramphenicol-resistant CFU will be counted on M17G plates with chloramphenicol. 
v.         Plasmid presence will be confirmed through plasmid extraction and amplification WHY DO YOU NEED TO “AMPLIFY” THE PLASMID DNA AFTER EXTRACTION (AND HOW WILL YOU DO IT)?. We will follow protocol demonstrated by the EDVOTEK Mini-prep isolation of plasmid DNA (7). 
 Data Collection and Data Analysis
We will initially collect data though the qualitative methods by observing growth, or lack thereof, via media platting and spectrophotometer. We will be analyzing the data using growth curve procedures. OD measurements from the spectrophotometer will be recorded with viable cell counts in a table; these will be plotted against each other forming a growth curve.  Images of gel electrophoresis will affirm presence or absence of the plasmid. All data will be input into Microsoft Excel and graphed using Microsoft Excel. We will test the results for statistical validity (p= <0.05) using   ???   (WHAT METHOD?)  of plasmid uptake, and differing growth under the simulated conditions within the rumen gut. 
Expected Results: 
We expect to see the wild type strain have an advantage in growth and replication over the vector strain of L. lactis, under the burden of expression of the NICE. This result is measured by using growth curves provided by observational methods and quantitative through the use of serial dilutions and plating on selective (antibiotic-supplemented media).  The unpredicted result of this experiment is a growth curve showing faster growth of the vector strain. While not likely, this outcome would be welcome in experimentation. This would mean that the vector strain would have a chance in competition with wild strains. We also expect to see presence of the plasmid to be diminished in non-selective environments, including the in-vitro rumen-like conditions.  CAN YOU QUALIFY THIS STATEMENT?  IE.  DIMISHED BY A LOT , OR A LITTLE.   ….DOES IT MATTER?
Timeline:
 
Week 0
Streak and isolate L. lactis (wild type, and transformed), Make M17 media with 0.5% glucose. 
 
Week 1
Test growth rates and abundance between the L. lactis wild type and transformed strain. Make M17 broth with 0.5% glucose. Grow L. lactis measuring the growth curve for four days, measuring every 12 hours using a spectrophotometer.  
 
Week 2
Test growth rates of the L. lactis wild type and transformed strain in the different pH, 5.7 and 7.3 by creating pH specific broth cultures. Growing the strains on a four-day growth curve. Measuring growth every 12 hours using a spectrophotometer. 
 
Week 3
Test growth rates of the L. lactis wild type and conjugated strain incubating in 40 degrees Celsius. Using a four-day growth curve, measuring density every 12 hours. 
 
Week 4
Determine the presence of plasmid through five generations of the L. lactis growth in M17 broth with 0.5% glucose. Plasmid presence will be confirmed by a plasmid extraction kit. 
 
Week 5
We will insert the naked plasmid THIS EXP. SHOULD BE LOW PRIORITY ;-) into M17 broth with 0.5% glucose of each of the following microbes: Salmonella enterica, Yersinia enterocolitica, Campylobacter jejuni, B. subtilis, and E. coli. Plasmid presence will be determined by streaking the pathogens on M17 0.5% glucose plates with chloramphenicol. 
 
Week 6
Colonies present on the chloramphenicol plates with growth will be run through a plasmid extraction kit to confirm plasmid presence, assuring growth was not a random mutation. 
 
Week 7
Data analysis using Microsoft Excel.
 
 
 
 
 
 
 
 
Sources Cited:[RW5] 
1.     “Brucellosis,” Ansci.wisc.edu http://www.ansci.wisc.edu/jjp1/ansci_repro/lab/diseases_2002/brucellosis.html 
 
2.     “Facts about Brucellosis,” Aphis.usda.gov, www.aphis.usda.gov/animal_health/animal_diseases/brucellosis/downloads/bruc- facts.pdf.
 
3.     J.E. Lowry, D.D. Isaak, J.A. Leonhardt, G. Vernati, J.C. Pate, G.P. Andrews, “ Vaccination with Brucella abortus Recombinant Induced Antigens reduces Bacterial Load and Promotes Clearance in a Mouse Model for Infection” PLoS ONE, 2011
 
4.     J. Neef, F.J. Milder, D.G.A.M. Koedijk, M. Klaassens, E.C. Heezius, J.A.G. van Strijp, A. Otto, D. Becher, J.M. van Dijl, G. Buist, “Versatile vector suit for the extracytoplasmic production and purification of heterologous His-tagged proteins in Lactococcus lactis,” Journal of Applied Genetics and Molecular Biotechnology, 2015(99)9037-9048.
 
5.     “M17 Agar.” CM0785, M17 Agar | Oxoid - Product Detail, 2017. www.oxoid.com/uk/blue/prod_detail/prod_detail.asp?pr=CM0785&org=82&.
 
6.     Found on http://www.edvotek.com/site/pdf/202.pdf
 
 [RW1]And why is this the best method to accomplish your goals?
 [RW2]It’s the viability of the bacterium that you are testing right? (e.g. we want to know if Lactococcus lactis is alive right?)
 [RW3]This is a confusing sentence, could we rephrase?
 [RW4]Can we return one more time to this section because there still seems to be an alignment issue with this and your now clear specific aims (above). (e.g. above we state that growth on selective media will be used to determine plasmid presence. I think that is the easiest way to do it. You can use PCR but it is more difficult.)
 [RW5]Re-add your annotations and recheck citation format.