1. Modifications of the Catalytic Properties of Butane Monooxygenase in P. butanovora Sarah Jean Mentors: Dr. Daniel J. Arp Dr. Luis Sayavedra-Soto Kim Halsey

2. Environmental Significance Pseudomonas butanovora metabolizes alkanes by oxidizing the alkanes to alcohols Alkane metabolism importance: 1.carbon cycling 2.bioremediation (fossil fuels and chlorinated aliphatic hydrocarbons such as chloroform and trichloroethylene) Bioremediation: Use of microorganisms to remove or detoxify toxic or unwanted chemicals from an environment. Trichloroethylene: (TCE) A volatile organic compound widely used as an industrial degreaser; as a solvent for oils, paints, and varnishes; and as a dry cleaning agent. Chloroform: Produced mainly by the chlorination of methane, chloroform is used in the production of refrigerants and agrochemicals. It is no longer used as an anesthetic.

3. Introduction to P. butanovora Uses a soluble diiron- containing monooxygenase to metabolize the alkane Structurally similar to methane monooxygenase (MMO), but can’t metabolize methane Monooxygenase: introduces one atom of dioxygen into the substrate and reduces the other atom to water C 4 H 10 + O 2 + AH 2  C 4 H 9 OH + H 2 O + A

4. Structural genes of BMO and MMO bmoXbmoYbmoBbmoZbmoDbmoC mmoXmmoYmmoBmmoZmmoDmmoC BMO reductaseMMO reductase BMO hydroxylaseMMO hydroxylase BMOB MMOB BUTANEMETHANE BUTANOLMETHANOL            hydroxylase,  hydroxylase,  hydroxylase,  reductase

5. Approach to modify BMOB Find out how BMOB determines what alkanes can be metabolized by BMO Create mutations in the DNA sequence coding BMOB Purify the modified BMOB WT BMOB mutant BMOB Y Y Y A

6. Then What? Test the effect of the BMOB mutations on sBMO in vitro and in vivo in P. butanova BMO reductase BMO hydroxylase WT BMOB Mutant BMOB METHANE ? METHANOL            

7. Procedure of purification Clone into pET- 15b, propagate in DH5-  E. Coli Plasmid Prep Transfer into BL21 E. Coli Grow to OD 600 0.6 Add IPTG & grow for 3 hours His-Bind purification dialysis Protein Quantification

8. DNA plasmid preparation Carry out the mutagenesis reaction Transformation DH5  Sequencing Determine concentration of plasmid DH5  plasmid preps

9. Methods: BMOB Expressions Transformation into BL21 IPTG LB media Expression to gel His-Bind purification column SDS-PAGE Purified protein After dialysis

10. BMOB Purification Progress Mutants of BMOB BMOB cloned into pET15B pET15B transformed into BL21 Expression of BMOB Purification of BMOB Dialysis of BMOB Protein quantification T109A T108S N105G P32H T108A T109Y H8A M114A Y100 frame shift V110A

11. Summary 8 BMOB mutations were purified and protein concentrations were determined for all of them Future research: Growth curves and activity assays will be carried out to see if changes in specificity were achieved

12. Acknowledgements Howard Hughes Medical Institute Dr. Daniel J. Arp Dr. Luis Sayavedra-Soto Kim Halsey Dr. Kevin Ahern Everyone else in the Nitrogen Fixation Lab Thank you for all the help!!