Cloning the
OOMT2 Gene
in Roses
Kim Lovik
Megan Hughes
Source Article
 Scent
evolution in Chinese roses
 Doi: 10.1073/pnas.0711551105
About the Gene
 Orcinol
O-methyltransferase 2
 Catalyzes the final methylation reaction of
DMT biosynthesis

DMT 3,5–dimethoxytoluene
A
major component of scent of many
modern rose varieties
DNA Source
 OOMT2
gene is common to all rose
species
 Plan is to use rose petals from a common
flower shop rose.

Although all parts of the rose contain the
gene, the production of the chemical
compounds is most prevalent in the petals.
Cloning Strategy

Amplify gene by PCR with short primers (without
BioBrick extensions)

Forward primer: ExonA1_F atggaaaggctaaacag


Reverse primer: ExonA2_R
tgcaatatcccttgagtaaaactgtatc


*no spontaneous dimer formation
Forward primer: ExonB1_F
aaatgagttcggatattgcatgactgg


*no spontaneous dimer formation
Spontaneous hairpin dimer formation ∆G -0.56 kj/mol*k
Reverse primer: ExonB2_R tcaaggataaacctcaat

Spontaneous hairpin dimer formation ∆G - 1.16 kj/mol*k

*Two sets of primers to remove intron from bp 799-1018
5’ atggaaaggc
atggaaaggc
aacgagctac
atgtccctca
atgactcttt
taccgcctca
actgatgagg
ttaagcctaa
tacttgagca
acattttggg
gctagcgatg
ggattagagt
gatgcattcc
caaggaagta
ctatgtcaaa
gatacagttt
atattatgta
caactattta
taaacag
taaacagctt
ttcatgctca
aatctgcaat
ctgagctcac
tgcgaatatt
aaggttatac
cgcccttctt
cttggttcca
attacgggaa
ctcggttggt
cattggtcga
cacatattga
agaacttgaa
atgagttccc
tactcaaggt
tatttcttct
aaacacaaag
taaacacctt aaccaaaaat
agcccacatc tggaaccaca
tcaactaggt ataccagata
atctgcacta ccaatccatc
ggtgcactct ggcttctttg
ccttactgat gcttcccagc
aaccgccatg ctcgaccctg
aaacgaggac cctacgccat
ccatcagcca agtattgccc
caccagcgtg atcatcgatg
tgttggaggt ggtacaggaa
atgcactgta cttgatctcc
atatactgga ggtgacatgt
tataacgt 5’
aattaaattt cagatataat
atatgctatt ctacttgtga
taacagtcac gtctatatct
ggtcaaatgg
tcttcagctt
tcatcaacaa
caaccaaatc
ctaagaaaaa
tccttctgaa
ttttgaccac
ttgacacagc
atttgttcaa
attgcaaagg
ctgtggcaaa
cacatgtggt
ttgaggcagt
agagcattcc
cataaactcc
acatggcccc
ccacagcgtc
gctgagtaaa
ggatcatccc
accatggaat
acatgggatg
tgatgccatg
agtgtttgag
ggctattgct
ggctgacctg
tcctcctgcc
tttatcctaa aaaagtttgg
ttaaaatgca ttattttagg
aattcactga ccaatataaa
5’a aatgagttcg
actctggacc aagaccaaag cacttctaat gcttatagtt attttatgtc acgagcagtg
gatattgcat gactgg
gatattgcat gactggaacg atgaagaatg tatcaaaata cttaagcgat ctagagtggc
aattacaagc aaggacaaga aaggcaaggt gattatcata gatatgatga tggagaacca
gaagggggat gaggaatcaa ttgaaacgca gctgttcttc gacatgctga tgatggccct
cgtcagagga caagaaagga atgagaaaga atgggctaag ctcttcactg atgctggttt
taactcc aaataggaac t 5’
cagtgactat aagataactc ccattttggg tttaaggtct ctcattgagg tttatccttg a
Cloning Strategy
 Insert
the amplified gene into
a pGEM-T vector via pGEM-T
and pGEM-T Easy Vector Systems
protocol
 Ligate back together
Check Progress
 The
DNA product inserted
in the vector will be amplified
using competent E.Coli
 Amplified product will be
tested to check for the OOMT2
gene.

Sequencing
Removing Internal Restriction
Site


One internal restriction site that would be cut by
XbaI (see next slide)
Site-Directed Mutagenesis used to mutate one
base pair using two new overlapping primers
One primer will contain a specified base pair
change, which will mutate the internal restriction
site. This will create two separate DNA fragments
cut near that mutated site.
The overlapping primers will be used much
like removing an intron, which will connect the two
DNA fragments back together.
Primers:
Check Progress
 Amplify
new DNA product after sitedirected mutagenesis, add XbaI
restriction enzyme to test results
 Use agarose gel electrophoresis to check
for proper gene length, to ensure the
fragments were reconstructed.
Insertion into BioBrick Vector
 Cut
out gene sequence from T-vector
with EcoRI and SpeI restriction enzymes
 Insert into BioBrick-compatible vector with
promoter



Bba_K206000-induced in the presence of Larabinose
Bba_K094120-induced by IPTG or arabinose
Bba_K206001-induced in the presence of Larabinose
Transformation
 Transform
competent E. coli with our
BioBrick vector containing our gene
fragment
 Provide conditions for gene expression

Proper growth conditions for E. coli in the
presence of arabinose for promoter
 Spread
E.Coli evenly on Prepared Agar Plates
 Grow overnight at 37°C
Results Test
 Add
3-methoxy 5-hydoxytoluene (MHT) to
the E.coli culture.
 MHT will be methylated by OOMT2
produced by the E.coli producing DHT
 Rose scent is produced by this chemical
reaction. It is described as “tea scent”
and is expected to be a noticeable
fragrance when the MHT is added to the
culture.