Tanner Dean and William McCleary, Microbiology and Molecular Biology

Introduction

Though Escherichia Coli is a thoroughly investigated species of bacteria, questions still remain concerning genes involved in phosphate homeostasis. Phosphate homeostasis is the idea that a cell or bacterium adapts to changing environmental phosphate concentrations. Cells need to adapt to changing phosphate conditions because phosphate is essential to the biochemistry of many cellular processes. A cell therefore, must be able to collect and store phosphate when there is excess or scavenge and utilize stored phosphate in times when there are low levels of extracellular phosphate available. This process is not entirely understood in E.coli or other single celled organisms. A new technique has been developed in the last two years to analyze essential genes and their respective fitness levels. This technique is called Transposon Sequencing (Tn- Seq). Tn-Seq involves creating a library of bacteria that have transposons randomly inserted into their genome. The random insertions will block certain genes from being expressed. The library can then be challenged under certain conditions. While the library is challenged, only the bacteria without essential genes blocked by the transposons will grow under those challenge conditions. We can then take those bacteria, sequence them and see which non-essential genes were blocked by transposons, and which genes were untouched by transposons and therefore essential to the phosphate conditions. This experiment can be performed on large scale so we can see which “essential” genes affect the fitness by using a computer program to analyze the sequences. This large-scale analysis will help us develop an idea of how each “essential” gene affects the fitness of E.coli under the challenge conditions. We will employ this technique to determine which genes are essential for E.coli to survive in high, medium and low phosphate conditions. We can then analyze the amount of the bacteria that have each “essential” gene and to what amount and make conclusions about how each gene affects the fitness under those challenge conditions.

Methodology

As Tn-Seq is a new experiment, my work was to develop the methods the lab will use to carry out these experiments. The first procedure was to develop a strain of E.coli that would be resistant to the antibiotic streptomycin. Streptomycin resistance was chosen because we needed an antibiotic that the strains used in the tri-parental mating weren’t resistant to. Streptomycin resistance arises from a single base-pair mutation in E.coli. To create the resistance we would grow bacteria on gradient media plates. The gradient media plates were used to select for the bacteria that grew at the highest concentration of streptomycin, eventually leading to a streptomycin resistant strain. We next tested our streptomycin resistant strain’s ability to grow on media of high, low and “normal” phosphate levels. This allowed us to develop an idea of how the strain interacts with the differing phosphate levels before exposure to the transposons and gene alterations. We next needed to develop the procedure for the tri-parental mating. We were given the basic steps from the Griffit’s Lab and then were set to work in fine-tuning the steps, conditions and other variables to create successful matings. A successful mating was one where the transformation resulted in approximately 300 colonies per plate. This step occupied the majority of the time. Once the conditions were solidified, we were then able to create a library of mutants that would have at least one transposon insert. The next step the lab will perform is to take mutants from the library and challenge them in media of our differing phosphate levels. They will then sequence the challenge media.

Note: Full step-by-step methodology will be available upon completion of the entire experiment.

Discussion

Sequencing the challenge media will reveal which unique transposon mutants were able to best colonize the specific challenge level of media. We can then compare that unique transposon mutant to the other transposon mutants and extrapolate which genes increase fitness under each condition. The conclusions drawn as to which genes may contribute to increased fitness in each condition will lead to further hypotheses, testing and results, almost like a treasure map. The treasure map of potential genes that increase fitness will not only give specifics into genes, but will also provide a clearer image as to what genes must all work together to allow E.coli to maintain phosphate homeostasis. Though I graduated before seeing this project to completion, there is a bright hope of gaining insight into the baffling question: how does phosphate homeostasis take place?