Dietz, Benjamin2023-02-032023-02-032022-10https://hdl.handle.net/11299/252317University of Minnesota M.S.B.A.E. thesis. October 2022. Major: Bioproducts/Biosystems Science Engineering and Management. Advisor: Brett Barney. 1 computer file (PDF); vii, 58 pages.Synthetically made nitrogenous based fertilizer has been relied on for decades to provide plants the nutrients they need to grow. Although this has helped increase the food production of crops, there has been some negative impacts of this as well such as high energy costs and leaching into water causing increased eutrophication. In order to reduce the impacts of synthetically made nitrogenous fertilizer biological nitrogen fixation has been an area of study. Recently, we have been interested in endophytic biological nitrogen fixation where the organisms would be able to deliver the nitrogen directly to the plant.In the first chapter we look at gene fitness during biological nitrogen fixation and non-biological nitrogen fixation. This was done through transposon mutagenesis where a transposon would insert itself into a TA site randomly disrupting the gene. This allowed us to make a large library of mutants to ensure sufficient coverage of all the TA sites. Once the library was made we grew it in the presence of nitrogen and without nitrogen forcing it to perform biological nitrogen fixation. We then sequenced the samples and analyzed gene fitness. This allowed us to understand what genes are essential or not essential during biological nitrogen fixation. This can potentially shed light on genes that are not previously known to be important for biological nitrogen fixation. It is also important when studying symbiotic endophyte relationships where the microbe fixes nitrogen for the plant. In the second chapter of this thesis we use what we know from other organisms that perform biological nitrogen fixation and are able to genetically modify a known endophyte Gluconacetobacter diazotrophicus so that there is an increase of ammonia/ammonium outside the cell. This could potentially lead to a better biofertilizer as it would supply nitrogen directly to the plant. In the third chapter we built a deoxyviolacein transposon insertion vector allowing us to quickly screen thousands of colonies for a preferred purple phenotype. The purple phenotype allowed us to distinguish the purple bacteria form other bacteria after they were infected in plants. We also believe that this vector will allow us to sense visually what the expression level of different genes are due to light and dark purple phenotypes. We also believe there are many different uses for this system beyond the scope of our lab.enThesis or Dissertation