Austin Jones Jace Dolphin

Methylosinus trichosporium culture courtesy of Dr. Alan DiSpirito, ISU Phenol/Chloroform Genomic DNA Extraction from protocol by Lab for Environmental Pathogens Research, U of Toledo Precipitated DNA was dissolved in water, used as template for PCR

 We were attempting to isolate a cluster of genes from the Methylosinus species of bacteria that enable them to breakdown and use methane for their energy and carbon source  Produce methane monooxygenase- oxidizes wide range of substrates  Saturated and unsaturated, linear, branched and cyclic compounds up to about C8, as well as aromatic, heterocyclic, and chlorinated compounds  Makes enzyme system ideal for petroleum spills, related cleanup

 Gene: Soluble Methane Monooxygenase (sMMO)  Accession # X55394  Amplify mmoX and mmoY separately from mmoB, mmoZ, mmoD, mmoC  Target genes to be ligated into two different vectors, both transformed into E. coli

 J62001 – Ampicillin-Resistant plasmid vector  pSB1K3 – Kanamycin-Resistant plasmid vector  J23100 – Constitutive Promoter Part  mmoXY – Target region of gene cluster, 2893bp  mmoBZDC – Target region of gene cluster, 2378bp

 Transformed J61002 plasmid (Ampicillin R., containing J23100 promoter), pSB1K3 (Kanamycin R.), and pSB1A7 (+control) into E. coli and plated  Plasmid DNA isolated from 2 colonies containing each plasmid for digestion

Original Idea – Remove J23100 promoter from J61002 backbone, to be ligated into pSB1K3. This way, end up with two vectors containing J23100, behind which target genes can be inserted. Digested J61002 with EcoR1 and Pst1 to remove, isolate insert and to confirm length of plasmid backbone – target 2103bp Digested pSB1K3 with EcoR1 to confirm length, linearize backbone – target 2204bp Digested pSB1A7 with EcoR1 – target 2431bp (+ control) J61002pSB1K3pSB1A7 C1 C2 Plasmids isolated from two colonies (C1,C2) each. Two elutions (lanes) per colony

Plasmids contained Red Fluorescent Protein (RFP) gene between biobricks 1022bp insert in pSB1K3 845bp insert in J62001 Also means J23100 that was ordered is unusable because of mixed biobricks site directly downstream

J23100 ACTAGT CTGCA 3’ TGATCA G 5’ 5’ CTAGA 3’ T SpeIPstIXbaI J23100 is small enough (35bp) to be ordered as an oligo set with biobrick sticky ends included Ordered two oligos that give this double strand when ligated 27F_J R_J23100 Included XbaI and PstI sticky ends for ligation to plasmid backbone Not self-compatible with XbaI and PstI sticky ends Performed a ligation to combine oligos

J61002 and pSB1K3 vectors digested with Xba1 and Pst1 Inserts separated from backbones on a gel pSB1K3 insert – 1022bp J61002 insert – 845bp pSB1K3 backbone – 2204bp J61002 backbone – 2103bp

Bands containing plasmid backbones were excised, DNA was purified Ligation was performed between digested plasmid (purified from gel) and assembled promoter (oligos w/ biobricks)

mmoBZDC 55˚ 60˚ 65˚ mmoXY 55˚ 60˚ 65˚ Target Product Sizes: - mmoXY: 2893bp - mmoBZDC: 2378bp

 Ligated J62001+J23100 and pSB1K3+J23100 parts were transformed into E. coli  No gel was run after ligation – J23100 too small to visibly change band size  Transformed cells plated on LB media  NO RESULTS  No colonies grew on antibiotic plates  Colonies present on +control plates, plates w/o antibiotics  Failed ligation?

 Ligation attempted again, but at 4˚C  Ligated promoter-plasmid parts again transformed into E.coli  RESULTS  2 colonies of pSB1K3+J23100 on kanamycin  Multiple colonies of J62001+J23100 on ampicillin  Grew colonies overnight in liquid LB for plasmid DNA extraction

5ul Template 1:10 Template Dilution mmoXY 45˚ 47˚ 50˚ mmoBZDC 45˚ 47˚ 50˚ - + Attempted again with: More template – 5ul Less template – 2ul of 1:10 dilution Lower annealing temperatures

 Cells containing pSB1K3+J23100 did not grow in liquid LB  Plasmid DNA isolated from 4 colonies presumably containing J62001+J23100  DNA sent for sequencing

 A03_J624_VR_ seq |TNNNNNNNNNNNNNNNNNN NNNNNNNNCNNNNNNNNNNNNNNNCNNNNNNNNG NNNNNNNGNNNNNNNNNNNNNCNNNNNTNNNNNN NNNNNGNNNNANTNTNTANNGNNNCTGGTNCNNNN NGNTTCCNNNNNGGANNGNNNNNNNTGNNCNCNNC GCANTNNANGNGANTNANNTNNNNNATTTNGNACCN CNNGNNTTNNNCNNTANGCTNNNNNNTCNNNTNNTG NGNGNAANNNNNNNNGNATNNNNNTNNCNCACNNG AAANNGCTNTGANCNTGANTNCNCCNAGNNNGCNNN TAANNCTCNNTANNNGGNANNNNANNTGGNNACNN NNNCCNCCCNCNANGNNNNCNGNNNNNATNNNNNN NNNNNNNNNTNNNNNCNNNNNGNNNNNNNNNNNN NNNNNNNANNNNNNNNNNNNNNNNNNNNNCTNCN NNNCNNCNNNNNNNNNNNNNNATNNNNNNNNNNC ANTGNNNNNNTTTTNNNNNNNNNNNNNNGNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNTNNNNNNNNNNNNTAANNNNNNNNN NNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNGNNNNTNNNNNNNNNNNNNNNNNNN

 The sequence we got back was mostly unreadable  Mess up?  Impure preparation?  Improper concentrations?  No time to do anything else

 Shigematsu, Toru, Satoshi Hanada, Masahiro Eguchi, and Yoichi Kamagata. " Soluble Methane Monooxygenase Gene Clusters from Trichloroethylene-Degrading Methylomonas sp. Strains and Detection of Methanotrophs during In Situ Bioremediation." APPLIED AND ENVIRONMENTAL MICROBIOLOGY (1999): NCBI. NIH, Dec Web. 27 Aug /am pdf /am pdf  Julie Scanlan, Marc G. Dumont, J. Colin Murrell Involvement of MmoR and MmoG in the transcriptional activation of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b. FEMS Microbiol Lett 301 (2009)  Genomic DNA Extraction Protocol from Univ. of Toledo: LEPR_Protocols_files/DNA%20extraction%20-%20culture.pdf LEPR_Protocols_files/DNA%20extraction%20-%20culture.pdf