Refactored Decaffeination Operon

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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.