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EXPRESSION OF METHANE MONOOXYGENASE IN E. COLI FOR BIODEGRADATION OF METHANE
Austin JonesJace Dolphin
Source Organism• 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
Purpose 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
Goals 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
Parts
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
Transformation
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
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)
J61002 pSB1K3 pSB1A7
C1 C2 C1 C2 C1 C2
Plasmids isolated from two colonies (C1,C2) each. Two elutions (lanes) per colony
Oh…
• 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
But…
J23100ACTAGT CTGCA 3’
TGATCA G 5’
5’ CTAGA
3’ T
SpeI PstIXbaI
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_J23100• 27R_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
Plasmid Digestion• 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
Gel Extraction & Ligation
Bands containing plasmid backbones were excised, DNA was purified
Ligation was performed between digested plasmid (purified from gel) and assembled promoter (oligos w/ biobricks)
PCR
mmoBZDC
55˚ 60˚ 65˚
mmoXY
55˚ 60˚ 65˚-- +
Target Product Sizes: - mmoXY: 2893bp - mmoBZDC: 2378bp
Transformation
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 & Transformation…2
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
PCR #2
5ulTemplate
1:10TemplateDilution mmoXY
45˚ 47˚ 50˚mmoBZDC45˚ 47˚ 50˚ - +
Attempted again with:• More template – 5ul• Less template – 2ul of 1:10 dilution• Lower annealing temperatures
Plasmid Extraction
Cells containing pSB1K3+J23100 did not grow in liquid LB
Plasmid DNA isolated from 4 colonies presumably containing J62001+J23100 DNA sent for sequencing
Results A03_J624_VR_803339.seq|
TNNNNNNNNNNNNNNNNNNNNNNNNNNCNNNNNNNNNNNNNNNCNNNNNNNNGNNNNNNNGNNNNNNNNNNNNNCNNNNNTNNNNNNNNNNNGNNNNANTNTNTANNGNNNCTGGTNCNNNNNGNTTCCNNNNNGGANNGNNNNNNNTGNNCNCNNCGCANTNNANGNGANTNANNTNNNNNATTTNGNACCNCNNGNNTTNNNCNNTANGCTNNNNNNTCNNNTNNTGNGNGNAANNNNNNNNGNATNNNNNTNNCNCACNNGAAANNGCTNTGANCNTGANTNCNCCNAGNNNGCNNNTAANNCTCNNTANNNGGNANNNNANNTGGNNACNNNNNCCNCCCNCNANGNNNNCNGNNNNNATNNNNNNNNNNNNNNNTNNNNNCNNNNNGNNNNNNNNNNNNNNNNNNNANNNNNNNNNNNNNNNNNNNNNCTNCNNNNCNNCNNNNNNNNNNNNNNATNNNNNNNNNNCANTGNNNNNNTTTTNNNNNNNNNNNNNNGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNTNNNNNNNNNNNNTAANNNNNNNNNNNNCNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN NNNNNNNGNNNNTNNNNNNNNNNNNNNNNNNN
Results, cont.
The sequence we got back was mostly unreadable Mess up? Impure preparation? Improper concentrations?
No time to do anything else
References
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 65.12 (1999): 5198- 206. NCBI. NIH, Dec. 1999. Web. 27 Aug. 2012.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC91705/pdf/am005198.pdf
Julie Scanlan, Marc G. Dumont, J. Colin MurrellInvolvement of MmoR and MmoG in the transcriptional activation of soluble methane monooxygenase genes in Methylosinus trichosporium OB3b. FEMS Microbiol Lett 301 (2009) 181-187
Genomic DNA Extraction Protocol from Univ. of Toledo: http://www.eeescience.utoledo.edu/Faculty/Sigler/Von_Sigler/LEPR_Protocols_files/DNA%20extraction%20-%20culture.pdf