Annealing temp 55C for 25 cycles, stored products at -20C
Made 3mL liquid cultures of the shipping vector (pSB1C3 with RFP insert) in DH5alpha in chloramphenicol resistant LB
Made 10mL liquid cultures of:
Topo in kanamycin
topo D168A in kanamycin
topo + GFPT1 in kanamycin + ampicillin
topo + GFPT2 in kanamycin + ampicillin
stored @ 37C
September 26
Assembled
Prsf Duest+Strep+L4+α which was a Success
Prsf Duest+Strep+L4+ω which was a Success
Prsf Duest+Strep+L+α which was a Success
Prsf Duest+Strep+L+ω which was a Success
Picked two new colonies for topo + GFPT2 and grew 10mL cultures of amp+kan LB broth for each
Ran a gel with Hyperladder I, GFPT1 pSB1A2, GFPT2 pSB1A2, Topo pSB1A2, Topo D168A
Ran a gel with PCR samples from 9/19 and 9/25 Hyperladder I, 1-1, 1-1I, 2-1, 2-1I, 2-1I(VF), 2-1(VF), 1-1I(VF), 1-1(VF), Hyperladder I
Samples in wells 2-5 used the pSB1A2 primer pair. Samples in wells 6-9 used VF2/VR primer pair.
Revived GFPT1/2 from glycerol stocks (from 8/9 and 8/15) in 5mL LB amp each.
Nanodropped miniprepped DNA samples:
pSB1C3 I - 253.75 ng/uL
pSB1C3 II - 258.38 ng/uL
Topo - 24.84 ng/uL
Topo I - 17.33 ng/uL
Topo D168A - 7.24 ng/uL
Topo D168A I - 15.62 ng/uL
1-1 - 16.49 ng/uL
1-1I - 16.46 ng/uL
2-1 - 142.05 ng/uL
2-1I - 16.98 ng/uL
Picked new colonies from Topo, Topo D168A, Topo + G1, Topo + G2 and made 1mL colonies in their respective medias
Stored at -37C
September 27
Testing R0011 promoter. Digested R0011 with S and P generated the following assembly:
R0011+Strep+L4+α which failed
R0011+Strep+L4+ω which failed
R0011+Strep+L+α which failed
R0011+Strep+L+ω which was a Success
Prepared serial dilutions of GFPT2 plasmid 1:10, 1:100, 1:1000, 1:10000
Prepared primer mixes for pSB and VF/VR primers
Prepared realtime PCR plate, ran RT-qPCR with annealing temp 57
Miniprepped GFPT1/2 cultures from 9/26
Nanodropped Miniprepped DNA:
GFPT1 - 276.12 ng/uL
GFPT2 - 219.84 ng/uL
Used 100uL of each 1mL culture from 9/26 to seed 10 mL cultures in their respective media
Added 10uL of 1M IPTG to each culture ~4 hours after seeding
Removed cells from 37C ~4 hours after IPTG inducing
Pelleted and lysed following the bugbuster protocol (http://openwetware.org/wiki/User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli/2012/05/22) (used lysonase for Topo and Topo D168A only)
HIS purified proteins using the Zymo HIS purification kit
Digested pSB1C3 plasmid with X and P
Sent protein samples (HIS purified samples from 9/19, 20uL) and ssDNA control (GFPT1/2 probe oligos 20uL @ 10uM) for mass spec
September 28
Tested another promoter (P2547) for the construct and repeated the assembly from 9/27.
Resuspended 8 new oligos, final volume 100 uM each
Annealed pet29 top/bot oligos
Redid the RT-PCR, 3x primer concentration, added 1:1 plasmid concentration
Made 1.6uM aliquots of primer stocks 1-7 (including topo add X primer previously ordered)
Diluted aliquot of Topo D168A plasmid 4:10
Diluted PSV plasmid 2:20
Performed Endpoint PCR on:
Topo D168A using primers 1,2 and primers 1,3
PSV using primers 4-5 and primers 6-7
4 samples with low primer concentration, 4 samples with twice as much primer (labelled 'H')
Miniprepped Topo1 2, Topo1 3, Topo1 4 (biobricked Topo D168A without T7, multiple colonies from ligation into shipping vector)
Nanodropped:
Topo1 2 - 63.63 ng/uL
Topo1 3 - 75.07 ng/uL
Topo1 4 - 184.93 ng/uL
1mL chloramphenicol cultures of GFPT1/GFPT2 in shipping vector prepared
Digested Topo1 2, Topo1 3, Topo1 4 with E and P
Ran digested Topo plasmids on 1% agarose gel with Hyperladder I
Revived cultures of 2xGFPT1, 2xGFPT2, Topo, and Topo D168A (4 mL cultures each in their respective medias)
Assembly was successful as liquid cultures were made. It was realized that due to the lack of beta gal negative strains the omega fragment couldn’t be tested and R0011 wouldn't code for the protein due to the lack of an RBS.
J61100 and J61101 constitutive promoters were extracted from the distribution plates and an additional promoter, pLux-Lac+RBS, were used to prove that the probe functions as desired.
Following assemblies were set up:
Plated on 50ul xgal LB amp Plates
J61100+ α
J61100+ strep+L+α (1a)
J61100+ strep+L+α (2c)
J61100+ strep +L +α(2b)
J61101+ α
J61101+strep+L+α(1a)
J61101+strep+L+α (2c)
J61101+strep+L+ α (2b)
plux-lac1+rbs+α
plux-lac1+rbs+strep+L+α(1a)
plux-lac1+rbs+strep+L+α(2c)
plux-lac1+rbs+strep+L+α(2b)
plux-lac2+rbs+α
plux-lac2+rbs+strep+L+α (1a)
plux-lac2+rbs+strep+L+α (2c)
plux-lac2+rbs+strep+L+α (2b)
Following were plated on 50ul X-Gal and 25ul IPTG LB AMP plates (no growth expected)
plux-lac1+rbs+α
plux-lac2+rbs+strep+L+α (1a)
plux-lac2+rbs+strep+L+α (2c)
plux-lac2+rbs+strep+L+α (2b)
Plate pictures:
Miniprepped GFPT1, GFPT2, Topo, and Topo D168A (Topo cultures separated into 3mL and 1mL minipreps)
Nanodropped miniprepped DNA:
GFPT1 I - 275.29 ng/uL
GFPT1 II - 101.08 ng/uL
GFPT2 I - 222.11 ng/uL
GFPT2 II - 230.52 ng/uL
Topo I - 117.97 ng/uL
Topo II - 70.28 ng/uL
Topo D168A I - 69.47 ng/uL
Topo D168A II - 51.87 ng/uL
HIS purified crude lysates from 9/27/12 (Topo, Topo D168A, Topo + GFPT1, Topo + GFPT2, all IPTG induced)
Digested Topo I plasmid (pet29a) with E and X
Ran a gel with Hyperladder I, 1-2 1-3 4-5 and 6-7 PCR products, pet29a digestion, and pSB1C3 digestion
Confirmed that PCR made amplicons
Excised bands for digested pet29a and pSB1C3 plasmids
Gel extracted 4 gel fragments (2 wells per sample: digested pSB1C3 plasmid, digested pet29a plasmid)
Nanodropped gel extractions:
Digested pSB1C3 - 25.54 ng/uL
Digested pet29a - 22.95 ng/uL
Set up a bradford assay of topo, topo D168A, topo + G1, topo + G2 (5uL protein, 10uL protein, 20uL protein + 200uL reagent)
Used ~100uL aliquot of BL21 competent glycerol stock to seed 10mL of LB medium (no antibiotic), stored at 37C
Ran 1% agarose gel with samples: A1, B1, A2, B2 and Hyperladder I
Did bug buster protocol to lyse BL21 control culture (used lysonase)
Ligated digested pet29a with pet29 top/bot annealed oligos
Transformed ligation using invitrogen DH5alpha transformation protocol
Prepared LB kanamycin plates
plated transformed ligations on prewarmed kanamycin plates
September 30
All assemblies were successful and went as planned. Blue colonies were picked to generate liquid cultures and streak plates to have a better visual result.
Pet29a plates did not grow
Kinase treated pet29a oligos
Annealed kinase treated pet29a oligos
Ligated digested pet29a (from gel extraction) with kinase treated oligos
Transformed ligations into DH5alpha, used topo plasmid as a positive control
Plated transformations on kanamycin plates and stored overnight at 37C
HIS purified BL21 control crude lysate
Set up a bradford assay with:
uninduced & induced topo protein extractions from 9/19
uninduced & induced topo D168A protein extractions from 9/19
BL21 control lysate
Topo, Topo D168A, Topo + G1, Topo + G2 from 9/27
All samples prepared (10uL protein, 20uL protein + 200uL reagent)
Miniprepped GFPT1 1,2,3 and GFPT2 (17,18,26) (~600uL of each) (1mL liquid cultures made from colonies on the chloramphenicol plates of GFPT1 and GFPT2 ligated into the shipping vector)
Nanodropped:
GFPT1-1 - 38.5 ng/uL
GFPT1-2 - 77.6 ng/uL
GFPT1-3 - 74.8 ng/uL
GFPT2-17 - 61.6 ng/uL
GFPT2-18 - 65.9 ng/uL
GFPT2-26 - 64.2 ng/uL
Ran a 1% agarose gel with 1-1, 1-2, 1-3, 2-17, 2-18, 2-26 plasmid miniprep samples and Hyperladder I
Prepared a 1:2 dilution of GFPT2 plasmid from 9/27 miniprep
Treated 5uL of diluted plasmid with 5uL of water, BL21 protein, topo protein, topo D168A protein
Incubated 30 minutes at 37C
Ran a 1% agarose gel with protein treated target plasmid samples and Hyperladder I
Digested GFPT2 plasmid with X (let run at 37C for 30 minutes)
Strep samples were minipreped and digested. The samples were inserted into the shipping vector.
Minipreps of:
J61011 + S + L + ALPHA 2B
J61100 + S + L + ALPHA 2B
J61100 + S + L + ALPHA 2C
PLUX2 + RBS + ALPHA 1A
PLUX2 + RBS + ALPHA 1A (2)
PLUX + S + L + ALPHA 2C
J61101 + S + L + ALPHA 1A
J61101 + S + L + ALPHA 1A (2)
PLUX2 + RBS + S + L + ALPHA 2B
PLUX2 + RBS + S + L + ALPHA 2B (2)
J61101 + S + L + ALPHA 2C
PLUX + S + L + ALPHA 2B
Ran Twice when finding concentrations:
J61101 + S + L + ALPHA 1A
J61101 + S + L + ALPHA 2B
Restricted all the above and:
Strep + Linker + OMEGA
Strep + Linker-4 + OMEGA
with:
X+P
Gel confirmed
Ligated into pSB1C3 shipping vector and transformed into BL21(DE3) cells.
Prepared all 14 above samples for sequencing.
October 2
Transformation failed and DNA was turned in for sequencing.
Prepared PCR tubes with 10uL Topo1 4 (25ng/uL topo d168a in the shipping vector), 10uL GFPT2-26 (25ng/uL GFPT2 in the shipping vector), and 10uL GFPT1-3 (25ng/uL GFPT1 in the shipping vector)
Labelled the tubes K891234, K891999, K891000 respectively and shipped overnight to iGEM Headquarters
Topo D168A treated DNA samples, incubated for 10 minutes at 37C:
Omega fragment PCR amplicon
Topo coding sequence PCR
GFPT1 VF2/VR PCR amplicon
Ran a 1% agarose gel containing untreated omega PCR, untreated topo PCR, untreated GFPT1 PCR, treated omega PCR, treated topo PCR, treated GFPT1 PCR, and Hyperladder I