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. This will be tested by growing the transformed strain in specific pH, temperature, and against wild-type and shuttle strains. 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 enteridisYersinia enterocoliticaCampylobacter jejuniEscherichia coli and Bacillus subtilis. Bacteria that acquire the NICE plasmid will be selected based on growth in chloramphenicol. We will select transformed colonies to extend over generations, testing the stability
 Materials and Methods:   
      i.         The laboratory of G.P. Andrews of the University of Wyoming will provide bacterial samples.
     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 30 degrees Celsius(6). We will also be streaking from frozen stalk onto TSA: Bacilis subtilis, E. coli. From frozen stalk onto blood agar plates (BPA) we will be streaking Salmonella enteridis, Yersinia enterocolitica, and Campylobacter jejuni (ADD CITATION FOR JEJUN 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 30 degrees Celsius. We will also streak the previously stated microbes for isolation for use in later tests. 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 titer using a spectometer. We will also use a similar procedure for testing the growth of L. lactis between the temperatures of 37.8 and 40 degrees Celsius. PCR will be performed using a standard PCR machine to look for plasmid presence in L. lactis after generations have been grown within the specific pH and temperature conditions3. We will also insert the naked plasmid into broth growths of the previously streaked stomach pathogens to test for the uptake of the plasmid. [RW3] We will also utilize plasmid extraction to determine the plasmids presence within the microbes (5).
iv. Serial subcultures will be performed by inoculating fresh medium 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. 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 evidence (p= <0.05) of plasmid uptake, and differing growth under the pressures within the rumen. 
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.  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 retainment of the plasmid to be diminished in non-selective environments, including the rumen-like conditions.