From 2012.igem.org
July 2012
S M T W T F S
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
August 2012
S M T W T F S
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
September 2012
S M T W T F S
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
October 2012
S M T W T F S
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
June 07
June 08
Transformation results
puc19: growth
negative control: no growth
lacZ, lacO: possible small colonies
liquid culture in amp media (100 ug / ml):
no growth of lacZ, lacO
growth of puc19
June 12
DH5a Competent Cell Prep
Streak plated cells on LB no amp plate, let grow overnight
June 13
Transformation (LSE)
Transformation (istb4, Abhi)
Transformed DNA:
Cells:
Protocol from:
Controls:
DH5a Chemically Competent cell prep
Grew 2 seed colonies from streak plate in LB no amp
Grew controls to test for contamination
Both Seed colonies grew, no contamination present
June 14
Competent cell prep
Prepared CaCl2 buffer solution and CaCl2 glycerol buffer solution
Grew seed colony in 400mL LB no amp
June 15
Competent cell prep
Centrifuged falcon test tubes containing liquid colonies
Resuspended in CaCl2 buffer solution and incubated for 15 mins
Centrifuged and resuspended in CaCl2 glycerol buffer solution
Chilled overnight
June 16
Competent cell prep
Aliquotted 200uL into test tubes
Stored in -80C
June 17
Streak plated prepared competent cells on LB no amp plate
June 19
Transformation (LSE)
Made 50 LB Amp plates.
June 20
Plated negative control on LB Amp plate
Liquid cultures of T7 promoter and constitutive promoter
Transformation (LSE)
June 21
Made Liquid Cultures of E.coli transformed with RBS B0030
Made Liquid Cultures of E.coli transformed with TetR GFP
miniprepped and nanodropped T7 promoter BBa_I712074 and Constitutive promoter BBa_J23102 liquid cultures
liquid cultures:
RBS1
RBS2 (duplicate_
GFP1
puc19
negative controls
5 ml LB amp
overnight cultures
replated GFP1 & 2 (duplicates)
Nanodropped plasmid DNA samples
Constitutive promoter 1: __ng/uL
Constitutive promoter 2: __ng/uL
T7 promoter 1: __ng/uL
T7 promoter 2: __ng/uL
June 22
Miniprepped liquid cultures: RBS (well 1:1H BBa_B0030) and TetR GFP (well 2:8A Part:BBa_I13522)
Picked colonies:
1 colony from double terminator (dt1) plate
1 colony from t7 polymerase (pol1) plate
1 colony from puc19 plate (positive control)
1 colony from dh5a plate (negative control)
started liquid cultures of each colony (5 mL LB amp each)
June 26
June 27
6-26 transformation results:
Controls correct
2x terminator: ~19 colonies
RNA pol: 1 colony
Liquid cultures including controls
June 28
Miniprepped double terminator (B0017, 2:6K) and T7 RNA polymerase (I715038, 2:15C) liquid cultures
July 2
Cleaned up liquid waste
Made SOB media
Finalized oligos for magainin construct
July 3
Autoclaved SOB media
Added glucose to make SOC media
Nanodropped double terminator (B0017, 2:6K) [DT1: 24.5, DT2: 29.6] and T7 RNA polymerase (I715038, 2:15C) [P1: 64.6, P2: 55.3] liquid cultures
July 24
Plasmids arrived courtesy of University of Pennsylvania School of Medicine
pET29a vectors containing coding sequence for Topoisomerase mutants CSCS and CSCS D168A described here
July 25
Tranformed CSCS topo 0 plasmid and CSCS D168A topo into DH5a Turbo cells (with neg control and Puc19 neg control)
Plated on Kanamycin plates
July 26
Picked colonies colonies from Topo 0 and Topo D168A and grew liquid cultures in Kan media
July 27
Miniprepped liquid colonies and nanodropped.
Plasmid concentrations
Topo O:
Topo D168A1:
Topo D168A2:
July 30
Prepared Kan Media and Kan Plates
July 31
PCR amplified polylinker sequence of Topo plasmid with Promega GoTaq protocol
Used pET29a upstream forward primer and T7 terminator reverse primer
August 3
Submitted pET29a Topoisomerase plasmid to Biodesign for sequencing
Resuspended GFPT1 and GFPT2 oligos with molecular grade (nuclease-free) H2O.
Final Concentration 100uM
(gfpt1 top1, gfpt2 top1, gfpt1 top2, gftp2 top2, gfpt1 bot1, gfpt2 bot1, gfpt1 bot2, gfpt2 bot2)
(3uL of each oligo + 2uL 10x annealing buffer, 6uL molecular grade H2O. 20uL Reactions)
Heated for 5 minutes at 100C. Let cool to room temperature on the heating block, stored at -20C.
Digested BBa_I13522 with XbaI and PstI.
Attempted ligating annealed oligos into a digested plasmid from Ryan (realized it was cut with E and P).
August 6
Annealed oligos for GFPT1 and GFPT2 (target probes)
Ligated oligos with digested GFP plasmid (BBa_I13522)
Transformed into competent DH5alpha
Added SOC and incubated at 37C for 15 minutes.
Plated on amp treated plates.
August 7
Only one colony on each plate (both were white)
Picked colonies and started 5mL LB amp cultures of each, stored at 37C
Stored plates in 37C
August 8
Picked the colonies again and started new liquid cultures (5mL LB amp).
Discarded cultures for 8/7/12
August 9
Miniprepped 3mL of each 8/8/12 culture and nanodropped:
gfpt1 - 155 ng/uL
gfpt2 - 114 ng/uL
Digested gfpt1 and gfpt2 with X and P
Ran on a 1% agarose gel with the digested GFP plasmid
Made glyercol stocks with aliquot of the remaining liquid cultures
August 10
PCR of Alpha-4, 1-omega, omega, and alpha fragments using corrected primers
August 13
Ran gel of split beta gal fragments. Confirmed 3 out of the 4 fragments except for the alpha-4 fragment.
Prepared sequencing samples
Sample w/ Primer:
GFPT1 w/ VF2 GFPT1 w/ VR GFPT2 w/ VF2 GFPT2 w/ VR
200ng of DNA + 16 pmol of primer
Annealed oligos again GFPT1/2
Repeated ligation of oligos with digested GFP plasmid (BBa_I13522)
Followed Haynes assembly protocol instead of standard DH5alpha protocol. (http://openwetware.org/wiki/Haynes:Assembly101 )
Transformed ligations into competent DH5alpha
Plated on amp treated plates
August 14
Took pictures of plates
Green-white screened plates
Picked 4 white colonies from each of gfpt1/2 plates
Made 5mL LB amp cultures of each colony
Delivered GFPT1/2 dna samples to biodesign for sequencing (samples from 8/13/12)
Assembled magainin insert via Overlapping oligo assembly
Digested pUC 19 plasmid with EcoRI and PstI
Transformed Magainin insert into digested pUC 19 plasmid. Failed. Probably too much X-gal on plate.
Ran gel for the beta gal alpha-4 fragment. Failed. Fragment not in the correct size-band.
August 15
Topo 0 , D168A Topo1 , and D168A Topo2 sequence results
Miniprepped 3mL of each liquid culture of GFPT1/2
Prepared glycerol stocks using 100uL of each liquid culture
Nanodropped samples:
GFPT1-1 - 172.6 ng/uL
GFPT1-2 - 203.7 ng/uL
GFPT1-3 - 197.4 ng/uL
GFPT1-4 - 178.9 ng/uL
GFPT2-1 - 107.3 ng/uL
GFPT2-2 - 131.2 ng/uL
GFPT2-3 - 145.5 ng/uL
GFPT2-4 - 172.0 ng/uL
August 16
Replated magainin insert + plasmid into the grid. Failed. All blue colonies meaning that no insert.
Tried gel for all gel-isolated fragments. Failed. Did not get a band in the 2000 bp region. Only got things below 200 bp.
August 17
GFPT1 sequence confirmed
Prepared aliquots of GFPT2 minipreps from 8/15/12 for sequencing
Delivered GFPT2 samples to biodesign for sequencing
August 19
GFPT2 sequences confirmed
August 27
Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes
Made 4mL cultures in LB Amp
August 29
Discarded GFPT1/2 cultures from 8/27/12
Revived GFPT1 (from 8/9/12) and GFPT2 (2-2 from 8/15/12) cultures from glycerol scrapes
Made 4mL cultures in LB Amp
Digested GFPT1/2 with X and S
Ran a 1% agarose gel with GFPT1/2 digestions
Cut out inserts and GFPT2 backbone and stored in 4C for gel extraction and tandum repeat assembly experiments
August 30
Prepared extra glyercol stocks of GFPT1/2 cultures from 8/29/12
Miniprepped 3mL of each culture, stored at -20C
September 19
Set up VF2/VR endpoint PCR for double transform minipreps
1-1, 1-1I, 1-2, 1-2I, 1-3, 1-3I, 2-1, 2-1I, 2-2, 2-2I, 2-3, 2-3I, GFPT1 (positive controls), GFPT2 (positive controls)
Annealing temp set to 55C for 25 cycles
Resuspended GFPT1 probe and GFPT2 probe oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C
September 25
Resuspended pSB1A2 FWD and pSB1A2 REV (amp resistance primers) oligos in molecular grade H2O (Final concentration: 100uM), stored at -20C
Prepared 1.6uM dilutions (500uL)
Did endpoint PCR using pSB1A2 primer pair on
Topo, Topo IPTG, Topo D168A, Topo D168A IPTG, 1-1, 1-1I, 2-1, 2-1I, GFPT1, GFPT2
Did endpoint PCR using VF2/VR primer pair on
Topo, Topo IPTG, Topo D168A, Topo D168A IPTG
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
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
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
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)
T5 exonuclease treated miniprepped plasmids (1-1, 1-1I)
A1 1-1I + t5
B1 1-1 + t5
A2 1-1I untreated
B2 1-1 untreated
September 29
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
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)
Used DNA clean up kit on digested GFPT2
Nanodropped digested GFPT2:
Prepared DNA seq samples using VF2 and VR
Sample# - PrimerPair - DNA sample (sample 1, GFPT2 uncut + VF2; sample 2, GFPT2 uncut + VR)
1/2 - FWD/REV - uncut GFPT2 plasmid
3/4 - FWD/REV - cut GFPT2 plamid
5/6 - FWD/REV - 2:1 uncut:cut GFPT2 plasmid mixture
7/8 - FWD/REV - 1:1 uncut:cut GFPT2 plasmid mixture
9/10 - FWD/REV - 1:2 uncut:cut GFPT2 plasmid mixture
11/12 - FWD/REV - 1-2 miniprep sample from 9/19 double transformations
13/14 - FWD/REV - 1-2I
15/16 - FWD/REV - 1-3
17/18 - FWD/REV - 1-3I
19/20 - FWD/REV - 2-1
21/22 - FWD/REV - 2-1I
23/24 - FWD/REV - 2-2
25/26 - FWD/REV - 2-2I
27/28 - FWD/REV - 2-3
29/30 - FWD/REV - 2-3I
October 1
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:
Ligated into pSB1C3 shipping vector and transformed into BL21(DE3) cells.
Prepared all 14 above samples for sequencing.
October 2
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