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

DigestionOriginal 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 pSB1A7C1 C2 C1 C2 C1 C2

Plasmids isolated from two colonies (C1,C2) each. Two elutions (lanes) per colony

Digestion Enzyme Test

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

mmoBZDC55˚ 60˚ 65˚

mmoXY55˚ 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