By Heidi Emmons and Dr. Kang Wu pbr Sporulation of B. subtilis for Heavy Metal Biosensors By Heidi Emmons and Dr. Kang Wu Sanborn Regional Middle School and Department of Chemical Engineering University of New Hampshire Discussion Introduction/Background Gel recovery shows DNA by making it glow under UV light. The ladder on the top indicates the number of base pairs to show if the DNA was the correct size. Gibson assemblies started to fail in the last research week and steps were taken to remedy the problem. Methods for troubleshooting included keeping DNA samples on ice during Gibson Assembly, new Gibson master mix was purchased, concentrations of DNA were measured, and incubation temperatures were monitored more closely. Heavy metals in the water and soil are problematic because they are difficult to detect and remove from the environment. Lead and mercury are heavy metals that often contaminate water and soil causing damage to animals and people even in small concentrations. In this experiment, different proteins are tested by first constructing DNA fragments and circular plasmids. The next phase is the integration of the DNA into B. subtilis for sporulation and heavy metal binding. These spores can be used as heavy metal biosensors because they have a thick coat making them resilient in varied environments. B. subtilis spores can be integrated with cotC-pbrR to bind heavy metals using a cell surface engineering approach. Next Steps Phase II - Integration of DNA into B. subtilis Linearization of DNA is necessary for B. subtilis transformation. The mini-prep method is used for this. Cells are spread on LB plates and tested using colony PCR. Phase III - Sporulation and lead binding testing of B. subtilis spores. Assembly G108-G121 joined JD9 with JD10-23 , which are CotC-PbrR(histag) and pbrRs of different types with and without histag. G122-135 assembled up amyE+cmR+cotC-pbrRs+down amyE. PCR used forward and reverse primers of different nucleotides to make more of the assembled DNA. There are many environmental applications for transformed B. subtilis spores as biosensors and for bioremediation because they are non-toxic and can be released into the environment. Literature Cited Isticato R. et al: Spore Surface Display Dept. of Bio. University of Naples, Naples, Italy Hobman et al: Cysteine coordination of Pb(II) is involved in the PbrR-dependent activation of the lead-resistance promoter, PpbrA, from Cupriavidius metallidurans CH34 BMC Microbiology 2012 12:109 Wu K. et al: Attractant Binding Induces Distinct Structural Changes to the Polar and Lateral Signaling Clusters in Bacillus subtilis Chemotaxis THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 4, pp. 2587–2595, January 28, 2011 Chen et al: Selective recognition of metal ions by metalloregulatory proteins ScienceDirect Borremans B. et al: Cloning and Functional Analysis of the pbr Lead Resistance Determinant of Ralstonia metallidurans CH34 JOURNAL OF BACTERIOLOGY, Oct. 2001, p. 5651–5658 Driks A.et al: The Spore Coat Dept. of Microbiology, Stritch School of Medicine, Loyola U. , Chicago, IL Isticaco R. et al: Surface Display of Recombinant Proteins on B. subtilis Spores Journal of Bacteriology  November 2001 vol. 183 no. 21 6294-6301 Background picture copied from Wikipedia B. subtilis spore with CotC-pbrR attached Methods Results There are three phases to complete the research plan. Phase I - Gibson assembly method to construct genes for the hybrid protein CotC-PbrR(s). Polymerase Chain Reaction (PCR) with the Gibson assembly as template. Gel electrophoresis to show DNA size. PCR clean up to purify the DNA (7.4 KB) Integration of linear DNA into B. subtilis. Heat shock transformation of circular DNA into E. coli cells. JD9 DNA was the first product to be tested using Gibson assembly to combine with JD10-23 and of the 13 tested, 5 showed DNA bands of the correct size on the gel. JD11, 14, 16, 17, and 20 were successful showing bright bands. The next DNA products tested were JD 24-37. Of the 14 samples, 10 showed very bright bands of the correct size on the Gel. JD24-26, 31-34, 36, and 37 were successfully assembled. Acknowledgements – This research was supported with funding from the National Science Foundation’s Research Experience for Teachers in Engineering Grant (ENG-1132648).