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Refactored Decaffeination Operon
From 2012.igem.org
<p>
Experiment 2: Constructon of a
refactored decaffeination operon
2a.
Phusion PCR
Primers:
EQ_pSB1c3_NdmA_for:
TAGGTACAGTGCTAGCTACTAGAGAAATCAAATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG
EQ_pBSC1C_NdmA_2:
TGATTTCTGGAATTCGCGGCCGCTTCTAGAGATTAAGGAGGTAAGATAAATGGAACAGGCAATCATTAATGATGAACGGG
EQ_pSB1C3_Gib_rev: TTGATTTCTCTAGTAGCTAGCACTGTACCTAGGACTG
EQ_NdmA_rev:
TTATATGTAGCTCCTATCGCTTTCAATGACTGGG
EQ_RBS_NdmB_for:
gtCATTGAAAGCGATAGGAGCTACATATAATCTAGAGAAAGAGGAGAAATACTAGATGAAAGAACAGCTCAAGCCGCTG
EQ_NdmB_rev: ccggtctcgcTTACTGTTCTTCTTCAATAACATTCGTCAAGACG
EQ_RBS_NdmC_for:
GAAGAAGAACAGTAAgcgagaccggTCTAGAGAAAGAGGAGAAATACTAGATGTCTACTGACCAAGTAATTTTTAACGactgg
EQ_NdmC_rev: TTAGTCCCGCAGAGCACCATATTGCac
EQ_RBS_NdmD for:
GCAATATGGTGCTCTGCGGGACTAATCTAGAGAAAGAGGAGAAATACTAGATGAACAAACTTGACGTCAACCagtgg
EQ_NdmD_pBS1C_rev:
TTATACAGCTCGTCCATACCGTGGGTGATGCCCGGCCTCACAGATCGAGAACGATT
EQ_pBS1C_Gib_for: GGGCATCACCCACGGTATGGACGAG
EQ_pBS1C_rev_2: CTCTAGAAGCGGCCGCGAATTCCAGAAATCA
10 uL 5x Phusion
HF Buffer
1 uL 10mM dNTPs
5 uL 5uM forward primer
5 uL 5uM reverse primer
1 uL DMSO
1 uL template
26.5 uL H20
--
.5 uL phusion
polymerase
rxns:
Vector PCRs
1. Bba_k515105 + EQ_pSB1C3_Gib_for +
EQ_pSB1c3_gib_rev
2. Bba_k515105 + EQ_pSB1C3_Gib_for +
EQ_pSB1c3_gib_rev_2
Insert PCRs
3. CBB5 cells + EQ_NdmA_for + EQ_NdmA_rev
4. CBB5 cells + EQ_pSB1C3_NdmA_2 + EQ_NdmA_rev
5. CBB5 cells + EQ_RBS_NdmB_for + EQ_NdmB_rev
6. CBB5 cells + EQ_RBS_NdmC_for + EQ_NdmC_rev
7. CBB5 cells + EQ_RBS_NdmD_for +
EQ_NdmD_pSB1C_rev
PCR cycling:
98 deg x 3 min
--
98 deg x 30s
58 deg x 20s x30 cycles
72 deg x 2 min
--
72 deg x 10 min
Gel igm2a
2b. PCR purification
Qiagen PCR purification protocol
Elute in 35 uL h20
nanodrop
concentrations:
1. vector 1: 289.0 ng/uL
2. vector 2: 85.6 ng/uL
3. NdmA_1: 139.8 ng/uL
4. NdmA_2: 144.5 ngl/uL
5. NdmB: 144.6ng/uL
6. NdmC: 146.1 ng/uL
7. NdmD: 104.8ng/uL
2c: DpnI digest of vector PCRs
1.
15 uL vector 1
5 uL 10x NEB 4 Buffer
1 uL dpnI
29 uL H20
--
50 uL
2.
30 uL vector 2
5 uL 10x NEB 4 Buffer
1 uL dpnI
14 uL H20
--
50 uL
37 deg x 2 hrs
2e. Purification
Standard Qiagen
PCR purification protocol. Elute
in H20.
2F. Gibson cloning
pmols =
weight(ng) x 1000 / (bp x
650 daltons)
Will use .05 pmol of vector, .1 pmol for each insert
Vec 1 (.05pmol) = need 68.57ng /
(79.5ng/uL) = .86uL
Vec 2 (.05pmol) = need 68.57ng /
(132.5ng/uL) = .52uL
NdmA1 (.1pmol) = need 71.5ng / (139.8ng/uL)
= .51 uL
NdmA2 (.1pmol) = need 71.5ng / (144.5ng/uL)
= .49 uL
NdmB (.1pmol) = need
73.4ng / (144.6ng/uL) = .50 uL
NdmC (.1pmol) = need
58.8ng / (146.1ng/uL) = .40 uL
NdmD (.1pmol) = need
120.ng / (104.8ng/uL) = 1.15 uL
rxns:
1.
.86 uL vec
1
.51 uL ndmA_1
.50 uL NdmB
.40 uL NdmC
1.15 uL NdmD
15 ul 1.33x Gibson Master Mix
1.58 uL H20
--
20 uL total
2.
.86 uL vec
1
15 uL 1.33x Gibson Master Mix
4.14 uL H20
--
20 uL total
3.
.52 uL Vec
2
.51 uL ndmA_1
.50 uL NdmB
.40 uL NdmC
1.15 uL NdmD
15 ul 1.33x Gibson Master Mix
1.94 uL H20
--
20 uL total
4.
.52 uL vec
1
15 uL 1.33x Gibson Master Mix
4.14 uL H20
--
20 uL total
50 deg thermocycler
x 60 minutes
Run 5 uL each reaction on .8% agarose gel
insert igm2f.jpeg
Desalted gibson
reactions on .025uM Nitrocellulose membranes x 20 minutes
2g.
Electroporation
Decided to not proceed with promoterless
construct (2F3,4)
1 uL desalted gibson
reaction (2F1,2) ->50 uL electrocompetent BW25113-GuaB -> 1mL SOC -> 1hr
incubation @ 37 deg
2j.
Selective growth in Caffeine/Theophylline
dilute 60 uL 2g transformations
(1-2) -> 3mL Mineral M9 media + 34ug/mL chloramphenicol +/- 100mg Caffeine
or Theophylline. Place on 30 deg shaker x 48 hrs
Results (growth):
2g1
(+operon) 2g2 (vector
only)
1. M9 - -
2. M9 + caffeine - -
3. M9 + Theophylline + _
Result: construct enables growth (demethylation)
on Theophylline. NdmC
is not fuctional.
Streak 2j1 theophylline enriched culture onto
LB-chloramphenicol plate for isolation of single colonies.
2K Plasmid Prep
Pick 2 colonies from theophylline enriched streak for
plasmid prep. Prep 5mL culture.
2L Restriction Digest
Perform restriction digest to analyze insert size of
plasmids
1 uL 2k1,2
1 uL 10x NEB 3
.5 uL NotI
6.5 uL H20
--
10 uL
37 deg x 1hr
Run 5 uL on .8% agarose gel
insert gel image (on darkroom
computer?)
Result: both clones
show anticipated ~5kB band corresponding to the complete operon.
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