Andrew Croteau1, Dr. Kang Wu2 Engineering the Sporulation Initiation Regulatory Network Andrew Croteau1, Dr. Kang Wu2 Department of Mathematics, The Founders Academy1 ,Department of Chemical Engineering, University of New Hampshire2 Introduction/Background Discussion Results Bacillus subtilis is a model bacterium to study sporulation, during which bacteria cells wrap the genetic materials with layers of protective proteins and form dormant spores in response to harsh environments. B. subtilis spores have been used as probiotics and also an excellent platform for protein display which has widespread applications from vaccine delivery to biosynthesis. Sporulation is initiated by processing the environmental signals through a complex network with Spo0A as the master regulator. In response to the external signal, Spo0A is gradually phosphorylated to Spo0A-P which can activate the expression of other essential proteins involved in sporulation. Using general protocols, about 10% of the cells sporulate. By inducibly expressing Spo0A and kinase that can phosphorylate it, the efficiency can be improved from 10% to 30%. The goal of this project is to systematically study the effect of the expression of all kinases and Spo0A on sporulation efficiency and then design a system to induce their expression by temperature. PCR’s that are highlighted were successful when running gel electrophoresis. Gel recovery was then performed afterwards. PCR Template: parent DNA, the DNA sequence we are interested in. Forward Primer: starting part DNA sequence Reverse Primer: end part DNA sequence DNTPs: reactant Polymerase: catalyst What could be causing Gel Electrophoresis to be unsuccessful? No DNA marker (ethidium bromide) Loading error (pipetting incorrectly) PCR not set up properly Low DNA quality (bands are faint or not visible) Too high of an electrical current. Future Work Gibson Assembly Diagram of the sporulation pathway  image of spores Successful Gels Methods Seed Culture → process of growing bacteria that will be experimented on Plasmid Mini-Prep → isolation of DNA Polymerase Chain Reaction (PCR) → copy segments of DNA Gel-Electrophoresis → separates DNA fragments based on their size Gel Recovery → isolate a desired fragment of DNA from an agrose gel Gibson Assembly → joins multiple DNA fragments Transformation Transfer DNA to E-coli Complete Mini-Prep to get desired plasmid Transfer plasmid to B. Subtilis Test multiple independent variables to analyze their affect on sporulation (temperature and pH levels) Size: 1.3 and 0.8 Kb PCR 4, 5 and 13 during gel recovery stage Size: 7.8 Kb (PCR 2) Size: 1.3 Kb (PCR 4 and 5) Acknowledgements Unsuccessful Gels This research was supported with funding from the National Science Foundation’s Research Experience for Teachers in Engineering Grant (ENG-1711781). I would like to Dr. Kang Wu, Guo Wu, Zhe Su and the Undergraduate Students who worked in the lab for assisting me in my work. I would also like to thank Dr. Stephen Hale, Allison Wasiewski and everyone who participated in the RETE program this summer for their help and guidance. Incorrect size band is present Not all bands are present. No bands are present Resources Note: Large molecules move slow through the gel, whereas small molecules move fast through the gel Ezio Ricca. 2014. Spore surface display.Microbiolspec 2(5): doi:10.1128/microbiolspec.TBS-0011-2012