Design

At the core of Northwestern’s Phytastic Probiotic project are two main subprojects.

The first is to clone different phytases from different organisms into E. coli and then to engineer E. coli to constitutively express the phytases at varying levels. We used a lab strain of E. coli as a model chassis for this project, but our technology could be adopted to function in probiotic strains as well. The four phytases we chose were phytases from E. coli, citrobacter braaki, aspergillus niger, and bacillus subtilus. These phytases were selected based upon varying specific activities, pH optima, and temperature optima that are expected to be most compatible with physiological conditions of the stomach, as compared to other phytases. Our general methods comprised PCR from genomic DNA, gibson assembly, and standard RFC10 biobrick assembly methods.

Construct

Each of the four phytases was PCR-amplified out of its respective organism’s genome. However, due to the introns present in the the Aspergillus niger phytase coding gene, we had an intron-free version of this gene synthesized by IDT. We assembled these parts together using Gibson assembly. After PCR cleanup, gel electrophoresis was performed to verify that the expected band patterns were observed.

After gel verifying and sequencing each of the phytases, an unexpected restriction site was discovered in the middle of the Bacillus subtilus phytase gene that prohibited biobrick assembly. Thus, we decided not to pursue this phytase construct further at this time. The three remaining phytases were cloned into protein expression backbones from the parts registry with constitutive promoters and ribosome binding sites already cloned into the vector. Several promoters from the same promoter library were used as each has varying relative expression strengths. Gel electrophoresis and sequencing was again performed to verify that the parts were assembled as expected.

Assay

To obtain a relative activity of the phytase produced by the cells with the constitutive promoter/citrobacter phytase part, we performed a phosphate assay on the produced phytase. In order to remove the phytase from the cells, the cells were overnighted and diluted 1:500 and then regrown into the end of log phase. Once at the end of log phase, the cells were centrifuged and sonicated in order to obtain the intracellular protein(with the produced phytase)to use in a phosphate assay. To perform the phosphate assay, .1 nmol phytic acid was added to the sonicated cell lysate at both a pH of 7 and a pH of 4.5. The pH of 4.5 was the literature value of pH that citrobacter phytase works best at. A colormetric phosphate assay solution was added and the OD of the solution was recorded in comparison to phosphate standards in order to obtain a measure of free phosphate in the solution.

From the above phosphate assay results, we concluded that the produced Citrobacter phytase grown in E. coli managed to liberate phosphate from the phytic acid solution. We are continuing work on performing more phosphate assays to obtain results with more statistical rigor.

As the regional jamboree approaches, we are continuing to obtain results for the phosphate assays performed on both the Aspergillus niger and E. coli phytase in order to better characterize our new phytase parts for the registry. Specifically , it would be beneficial to know which phytase is the best at liberating phosphate and iron from phytic acid. As each phytase has a different active pH range, optimum temperature, and specific activity we expect that each phytase will have drastically different results in the physiological conditions of the stomach. Our best guess of which phytase will perform the best in the conditions of the stomach is the Citrobacter braaki phytase, due to its high specific activity[1] and low pH optimum. [1] http://www.ncbi.nlm.nih.gov/pubmed/17302159