Team:Duke/Notebook/July

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May 2014
Month 2 of Project
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June 2014
Month 3 of Project
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July 1

Objective: make LB+Cm plates

  • Four sleeves of plates with 4x500mL medium
  • 34mg/mL chloramphenicol

Objective: insert crRNAs into pdCas9 and scaffold

        Plate results

  • Experimental had lots of colonies, but backbone-only control did as well
  • reasoning: similarity of BsaI overhangs may lead to re-ligation
  • solution: treat with CIP before ligation

Agarose gel of BsaI cuts of pdCas9 and Repeat scaffold

  • plasmids used in 6/30/14 transformation
  • to make sure BsaI is cutting properly
  • Results: single band in both appears to be cut DNA

        CIP treatment of pdCas9 and Repeat scaffold (both BsaI cut)

  • pdCas9: 10uL plasmid in 20 uL reaction with 1.5uL CIP
  • Repeat scaffold: 22uL plasmid in 30 uL reaction with 2 uL CIP
  • 2 hours at 37C (longer than standard protocol)

        

        PCR cleanup of pdCas9 and Repeat scaffold CIP treatments

  • Final concentrations too low to be of use
  • Particularly negligible in pdCas9
  • Need to culture and cut more plasmid to try ligation again

Spread plates from pdCas9 and Repeat scaffold frozen stocks

  • Used tube “1” for repeat scaffold
  • Plated on fresh LB+Cm plates
  • Plates still slightly wet, difficult to spread evenly

Objective: make CCEC stock of DH5alpha-ZI strain

        Diluted back 5mL culture in morning

  • 1mL into 5mL LB+Spec
  • note for future: Spec not necessary for most procedures, chromosomally integrated genes are more stable

        Inoculated 1 mL culture into each of two 250mL flasks

  • Flasks contain SOC
  • 30C shaker for overnight growing

July 2

Objective: Make CCEC stock of DH5alpha-ZI strain

        Made fresh CCMB-80 buffer stock

  • precipitate formed during pH adjusting and may have filtered out
  • could be detrimental to final product

        Followed iGEM online protocol to make CCEC from Z1 strain

  • Overnight culture was much more concentrated than recommended
  • OD >1
  • Did not have enough SOC to dilute back fully, so diluted back only a bit
  • Had to vortex to resuspend cells at two stages of procedure
  • Final OD after procedure ~1.6 for 100mL of cells
  • Aliquoted 1mL into tubes, froze at -80C

        Next steps:

  • Test competence with useful strains containing Lac, Tet promoters

Objective: insert crRNAs into pdCas9 and Repeat-scaffold

        Culture colonies from plates of pdCas9 and pSB1C3-Repeat-scaffold

  • 3 colonies per plate in LB+Cm medium at 37C overnight
  • Plates were messy--hard to get individual colonies

July 3

Objective: insert crRNAs into pdCas9 and Repeat-scaffold

        Miniprep cultures from 7/2

  • Three tubes of pdCas9 with concentrations 143.4, 145.7, 151.4 ng/uL
  • Three tubes of Repeat-scaffold with concentrations 166.6, 145.8, 159.6 ng/uL

        Digest tubes 1 and 2 of pdCas9 and tubes 1 and 2 of Repeat-scaffold

  • to insert crRNAs
  • Digest with BsaI in cutsmart buffer
  • 50 uL DNA in 100 uL reactions with 7.5 uL BsaI
  • 3 hours 20 min at 37C

        PCR cleanup of BsaI digest

  • final concentrations (in 30 uL each):
  • pdCas9: 65.4, 70.5 ng/uL
  • Repeat-scaffold: 122.3, 114.8 ng/uL

CIP treatment of pdCas9 and Repeat-scaffold BsaI digests

  • Two digest tubes pooled into one CIP tube for each backbone
  • 60 uL DNA in 100 uL reactions with 5 uL CIP in cutsmart buffer
  • 1.5 hours at 37C

        PCR cleanup of CIP treatment

  • final concentrations (in 30 uL):
  • pdCas9: 32.5 ng/uL
  • Repeat scaffold 129.9 ng/uL

Annealed oligos of crRNA inserts for ligation

  • 3 crRNAs (GFP1, GFP2, GFP3) with 2 oligos each
  • 5uL each oligo heated to ~95C, slowly brought to rt in water bath

        Ligation of crRNA inserts into pdCas9 and Repeat-scaffold

  • 6 working tubes + 5 controls
  • pdCas9 + each crRNA (GFP1, GFP2, or GFP3)
  • Repeat-scaffold + each crRNA (GFP1, GFP2, or GFP3)
  • pdCas9 Backbone only control
  • Repeat-scaffold Backbone only control
  • Insert only controls for each insert
  • 100ng = 3uL pdCas9, BsaI digested and CIP treated and cleaned
  • 100ng = 0.8uL Repeat-scaffold, BsaI digested and CIP treated and cleaned
  • 1uL annealed oligo insert
  • 10 uL reactions with 0.5 uL T4 Ligase
  • Transformed into DH5alpha and plated on LB+Cm

Objective: Test competency and effectiveness of DH5alpha-Z1 strain

        CCEC Transformation efficiency test

  • Transformed 5 tubes of pSB1C3-K741002 into DH5alpha-Z1 CCEC
  • Total amount of DNA in 10 uL: 130ng, 13ng, 1.3ng, 0.13ng, and 0.013ng
  • Also for use in testing pLac regulation in Z1 strain

        Transformed iGEM kit plate 3-6G into DH5alpha-Z1

  • contains pSB1C3-BBa_I13521: pTet-RBS-mRFP
  • in order to test regulation of pTet in Z1 strain

July 4

Objective: ligate crRNAs into pdCas9 and Repeat-scaffold

        Plate results for ligation of crRNA GFP1, GFP2, and GFP3 into two backbones:

  • ~1000 colonies on all experimental plates
  • BO control for pdCas9 has ~500 colonies
  • BO control for Repeat scaffold has ~1000 colonies
  • IO controls all have 0 colonies

        Cultured 4 copies of each crRNA-GFP1,2,3 in pdCas9 backbone

  • 12 tubes total in LB+Cm
  • None from Repeat-scaffold attempts

Objective: Test competency and effectiveness of DH5alpha-Z1 strain

        CCEC Test plate results:

  • 130 ng: 100s of colonies
  • 13 ng:~100 colonies
  • 1.3 ng: ~20 colonies
  • 0.13 ng: 0 colonies
  • 0.013 ng: 0 colonies
  • Competency ~10 colonies/ng (low but working)

        Results of transformation of pSB1C3-BBa_I13521 into DH5alpha-Z1

  • Plate had ~5 colonies

        Cultured colonies for Tet and Lac testing in DH5alpha-Z1

  • One colony of pSB1C3-K741002
  • One colony of pSB1C3-BBa_I13521
  • In LB+Cm

July 5

Objective: Insert crRNAs into pdCas9 backbone

        Saved stocks of 12 strains in 15% glycerol at -80C

  • 4 tubes each of potential GFP1,2,3 crRNA inserts in pdCas9

        Miniprepped 12 tubes of pdCas9 with potential GFP-1,2,3 inserts

  • 4 tubes each
  • Eluted in 50 uL EB, all concentrations 100-200 ng/uL

Objective: Test effectiveness of DH5alpha-Z1 strain

        Saved stock of pSB1C3-K741002 in DH5alpha (15% glycerol at -80C)

  • Culture of I13521 did not grow

        Cultured another colony of pSB1C3-BBa_I13521

  • from 7/3 plate

July 6

Objective: Test effectiveness of DH5alpha-Z1 strain

        Saved stock of pSB1C3-I13521 in DH5alpha (15% glycerol at -80C)

July 7

Objective: insert crRNAs GFP1,2,3 into pdCas9

        Analytical restriction digest

  • 12 tubes with potential inserts (4 per crRNA)
  • pdCas9 as a control
  • BsaI and XbaI in cutsmart for 1.5 hrs

        Agarose gel of restriction digest

  • Gel 1:
  • 1-4: pdCas9-crRNA-GFP1
  • 5-8: pdCas9-crRNA-GFP2
  • 9-12: pdCas9-crRNA-GFP3
  • Gel 2:
  • pdCas9 (control)
  • Expected single band ~9.3 kb if BsaI cut and ligation worked
  • Control and uncut failures expect two bands, 5.0 and 4.2 kb
  • Results: all 12 samples have single band
  • No uncut plasmids
  • Could still be recircularized with no insert

gel pictures:

        Anneal oligos of 3 crRNA-GFP inserts

  • Following Dewran’s crRNA protocol
  • 2 uL each oligo diluted in PBS to 20 uL
  • Placed in 98C water bath, cooled gradually to rt

        Phosphorylate oligo inserts of crRNAs

  • 5 uL annealed oligo mix in 20uL 1x ligase buffer with 1uL PNK
  • 30 mins at 37C, then deactivated for 20 mins at 65C
  • Diluted 1/50

        Ligation

  • 75 ng backbone = 0.6 uL Repeat scaffold or 2.5 uL pdCas9
  • Both BsaI cut and CIP treated 7/3/14
  • 1.5 uL diluted, phosphorylated, annealed oligo insert
  • 6 experimental tubes + 2 BO controls and 3 IO controls
  • Transformed into iGEM CCEC (DH5alpha) and plated on LB+Cm

        Cultured 2 colonies from pdCas9 plate

  • For Charlie to try his hands at ligation

Objective: Prepare various backbones for multiple-plasmid use

        Transformed two distribution kit parts

  • Plate 4-6H: pSB4K5-J04450
  • backbone contains pSC101 origin with Kan resistance
  • Plate 4-18A: pSB3K3-I20260
  • backbone contains p15A origin with Kan resistance
  • On LB+G418 plates

Streaked plate from frozen stock

  • pSB6A1-J04450
  • backbone contains pMB1 origin with Amp resistance
  • on LB+Amp

Objective: Test pTet and pLac in DH5alphaZ1 strain

        Streaked plates from frozen stock

  • pSB1C3-K741002 in DH5alpha-Z1
  • contains pLac-GFP
  • pSB1C3-I13521 in DH5alpha-ZI
  • contains pTet-RFP
  • On LB+Cm plates

July 8

Objective: insert crRNAs into pdCas9

        Plate results from 7/7 transformation

  • pdCas9-crRNAs: ~50 colonies each
  • pdCas9 BO: 100s of colonies
  • Repeat-crRNAs: 100s of colonies
  • Repeat BO: ~1000 colonies
  • IO controls: 0 colonies, ~15 for crRNA-GFP3
  • Notes: unsure why BO controls have more colonies than experimental

        Restriction digest of 12 samples from 7/5 prep + pdCas9 control

  • With SacI/NheI
  • Should show difference between insert and recircularized backbone

        Made and ran 1.5% agarose gel in TBE

  • for better resolution of small band differences in pdCas9 with/without insert
  • 1: pdCas9 (control)
  • 2-5: pdCas9-crRNA-GFP1 (1-4)
  • 6-9: pdCas9-crRNA-GFP2 (1-4)
  • 10-13: pdCas9-crRNA-GFP3 (1-4)
  • 14: pdCas9 (control)
  • expected:
  • 3.1, 2.8, and 2.7 kb bands
  • 585 bp band with no insert, 620 bp band with insert (pdCas9 620bp)
  • Results: One sample, crRNA-GFP3-3, has larger band matching control
  • This colony may be correct, should be sequenced
  • Others have smaller band, are probably incorrect

Culture 40 colonies from existing plates to screen for crRNA inserts

  • 10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/3/14
  • 10 each from crRNA-GFP1 and crRNA-GFP2 transformed 7/7/14
  • in LB+Cm

Objective: Prepare various backbones for multiple-plasmid use

        Prepared LB+Kan medium

  • 50 ug/mL final concentration Kanamycin

        Cultured colonies from transformations and stock streaking

  • 3 colonies each from 3 plasmids:
  • pSB6A1-J04450 in LB+Amp
  • pSB3K3-I20260 in LB+Kan
  • pSB4K5-J04450 in LB+Kan

Objective: Test pLac and pTet in DH5alpha-Z1

        Notes:

  • Need to obtain aTc for pTet testing
  • Tried to use Doxycycline, but had trouble with solubility in ethanol (?)
  • pLac is not the final promoter to be used, a different variant will be used
  • with no glucose dependency
  • Flow testing of pLac and pTet was suspended until these changes are made

July 9

Objective: Insert crRNAs into pdCas9

Miniprep 40 cultures of potential crRNA inserts in pdCas9

  • Labeled with three numbers
    • Date of transformation - crRNA-GFPx - sample number

700uL of each culture saved to freeze in glycerol if colonies appear successful

  • Stored at 4C for the day

Analytical restriction digest of potential crRNA inserts

  • All 40 tubes + 2 pdCas9 tubes as control
  • 1 uL DNA in 10 uL digest for 2 hrs at 37C
  • SacI-HF and NheI-HF in cutsmart

Made and ran 1.5% agarose/TBE gels

  • To differentiate between small band differences
  • Gel 1:
    • 1. 2-log ladder
    • 2. pdCas9
    • 3-12. pdCas9-crRNA-GFP1 from 7/3 transformation
    • 13-22. pdCas9 -crRNA-GFP2 from 7/3 transformation
    • 23. pdCas9
    • 24. 2-log ladder
  • Gel 2:
    • 1. 2-log ladder
    • 2. pdCas9
    • 3-12. pdCas9-crRNA-GFP1 from 7/7 transformation
    • 13-22. pdCas9 -crRNA-GFP2 from 7/7 transformation
    • 23. pdCas9
    • 24. 2-log ladder
  • Expected results:
    • pdCas9 and successful inserts:
      • 3.1, 2.8, and 2.7 kb bands, plus 620 bp insert band
    • unsuccessful recircularizations:
      • 3.1, 2.8, and 2.7 kb bands, plus 585 bp insert band
  • Results:
    • Gel 1 appears to only be recircularized
    • Gel 2 has some promising inserts:
      • GFP1 samples 1,2, and 3 from 7/7 transformation
      • GFP2 samples 7,8, and 9 from 7/7 transformation
  • Glycerol stocks frozen for these six samples
  • Next Steps:
    • Run digest with BsaI to confirm that plasmids are not uncut pdCas9
    • Sequence to confirm presence of insert (using oligos ordered 7/8/14)
Objective: Obtain new backbones and switch inserts into new backbones

Miniprep 3 new backbones

  • pSB6A1-J04450 (3 copies)
    • concentrations 437.6, 483.9, and 450.1 ng/uL
  • pSB3K3-I20260 (3 copies)
    • concentrations 114.6, 120.0, and 97.3 ng/uL
  • pSB4K5-J04450 (3 copies)
    • concentrations 129.3, 165.9, and 155.1 ng/uL

Prep-scale digest of all 9 backbone tubes

  • 50 uL DNA in 60 uL total reaction
  • EcoRI-HF/Spe-HF in cutsmart
  • 3 hours at 37C

Agarose gel extraction of 3 backbones

  • Gel 1, Left: pSB6A1
  • Gel 1, Right: pSB3K3
  • Gel 2, Left: pSB4K5
  • All 3 lanes had 2 bands as expected
    • upper band (backbone) in 3K3 had messy trail behind it (not extracted)
  • All 180 uL from three tubes combined with 20 uL loading dye in x-large gel well
  • top band extracted from all 3 plasmids, split into 2 tubes each, stored at -20C

Streak plate from frozen stock

  • pSB1C3-K608012
  • In order to switch into pSB6A1
Objective: Remake new version of pDGC3

Transformation from iGEM distribution kit plates

  • Plate 2-6D: pSB1C3-BBa_R0011
    • Engineered Lac promoter (not affected by glucose)
  • Plate 3-15O: pSB1C3-BBa_I13500
    • Strong RBS-GFP
  • Plated on LB+Cm

July 10

Objective: Obtain backbones and switch parts into new backbones

Gel cleanup of 3 backbones with Zymoclean prep kit

  • Some tubes had to be divided into two samples for cleanup
  • Final elution was 20 uL each into 2-3 tubes per backbone
  • Nanodrops looked unclean: Most had high peak around 220-240 nm
    • pSB6A1: 300mg + 120 mg gel = 230.4 ng/uL
      • Peak ~220, then hump ~260
    • pSB6A1: 270 mg gel = 220 ng/uL
      • Peak, then hump as before
    • pSB3K3: 320 mg gel = 53.9 ng/uL
      • No clear sign of DNA in graph
    • pSB3K3: 230 mg gel = 20 ng/uL
      • No DNA
    • pSB3K3: 130 mg gel = 72 ng/uL
      • Possibly DNA, best option but may not be useful
    • pSB4K5: 290 mg gel = 43.4 ng/uL
      • No clear sign of DNA in graph
    • pSB4K5: 320 mg gel = 117.3 ng/uL
      • Possibly DNA, best option but may not be useful

Culture 3 colonies of pSB1C3-K608012

  • In order to switch into pSB6A1
Objective: Insert crRNAs into pdCas9

Analytical Digest of promising crRNA inserts

  • XbaI/BsaI digest
  • In order to confirm that plasmids are not uncut pdCas9
  • Samples 7-1-1,2,3 and 7-2-7,8,9 (6 tubes) plus pdCas9 as control

Agarose gel of digest results:

  • 1. pdCas9
  • 2-4. pdCas9-crRNA-GFP1, samples 1,2,3
  • 5-7. pdCas9-crRNA-GFP2, samples 7,8,9
  • Expected:
    • 2 bands at 4.2 and 5.1 kb in pdCas9
    • 1 band at 9.3 kb in successful plasmids with inserts
  • Results:
    • pdCas9 appears to be an incomplete digest, showing 2 small and 1 large band
    • Lack of any trace of smaller bands in 6 experimental samples indicates successful inserts.
Objective: Make pDGC3

Culture 3 colonies each from pSB1C3-R0011 and pSB1C3-I13500

Objective: Test effectiveness of DH5alpha-Z1 strain
Mitch and Sargis

Culture pSB1C3-I13521 (pTet-RFP)

  • For flow
  • In aTc and non-aTc

July 11

Objective: Switch K608012 into pSB6A1

Miniprep 3 copies of pSB1C3-K608012

  • concentrations 361, 324, 362 ng/uL

Prep-scale digest of pSB1C3-K608012

  • 2 miniprep tubes, 50 uL DNA in 100 uL each
  • With EcoRI and SpeI
  • 2+ hrs at 37C

Gel extraction and cleanup to isolate K608012

  • Cut lower band
  • Zymoclean prep kit
  • Each of 2 bands divided into two tubes during cleanup
    • 1A: 280 mg =
    • 1B: 240 mg =
    • 2A: 200 mg =
    • 2B: 200 mg = 62.5 ng/uL (20 uL)
  • Graphs on nanodrop did not look ideal: peak lower than usual
Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block

Miniprep pSB1C3-R0011 and pSB1C3-I13500

  • 3 copies each
  • Concentrations
    • pSB1C3-R0011: 181, 229, 194 ng/uL
    • pSB1C3-I13500: 321.5, 228, 300.4 ng/uL

Prep scale digests of two tubes from each miniprep

  • pSB1C3-R0011 with SpeI/PstI
    • no extraction necessary
  • pSB1C3-I13521 with XbaI/PstI
    • to extract I13521 insert
  • 100 uL total reaction per tube
  • 2-3 hrs at 37C

Agarose gel of pSB1C3-I13500 XbaI/PstI digests

  • Intended for gel extraction
  • Expected 2 bands, but only one present
  • No extraction performed

Analytical agarose gel of pSB1C3-R0011 SpeI/PstI digests

  • Single band appears correct
  • see below for gel picture

PCR cleanup of pSB1C3-R0011 SpeI/PstI digests

  • concentrations 124.8, 157.3 ng/uL (30 uL each)
Objective: Switch dCas9-tracrRNA into pSB1C3

Miniprep pdCas9

  • concentration 167.4 ng/uL

Prep-scale digest of pSB1C3-R0011

  • 1 miniprep with XbaI/PstI
  • To extract pSB1C3 backbone

Agarose gel of pSB1C3-R0011 XbaI/PstI digest

  • Intended for gel extraction
  • Expected 2 bands, but only one present
  • No extraction performed

PCR of dCas9-tracrRNA and anti-tracrRNA

  • 4 tubes each, with pdCas9 as template in 0, 0.4, 0.7, and 1.0 uL quantities
  • oligos dCas9tracr-up and dCas9tracr-dn for dCas9-tracrRNA PCR
  • oligos dCas9tracr-up and tracrRNA-dn for anti-tracrRNA PCR
  • Q5 polymerase, with 64C annealing and 2 min extension

Agarose gel of PCR (and digest) results:

  • Lanes 1-4: dCas9-tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
  • Lanes 5-8: tracrRNA PCR (0, 0.4, 0.7, 1.0 uL template)
  • Lanes 9-10: pSB1C3-R0011 SpeI/PstI digests
  • Results:
    • dCas9-tracr PCR appears correct, band ~4 kb in 3 lanes
    • tracrRNA PCR unclear: strong primer-sized band may be correct, faint bands ~4 kb indicate off-target extension.
      • tracrRNA PCR should be done with shorter extension time

PCR cleanup of dCas9-tracrRNA PCR

  • Concentration >200 ng/uL
Objective: Test BsaI and XbaI enzymes

Analytical digest of dCas9

  • One tube with BsaI/EcoRI
  • One tube with XbaI/EcoRI
    • XbaI from tube with exp. 5/14
  • One tube with XbaI/EcoRI
    • XbaI from tube with exp. 2/15

Agarose gel of digest:

  • 1. BsaI/EcoRI: expected 3 bands at 2.7, 2.9, and 3.5 kb
  • 2. XbaI (5/14) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
  • 3. XbaI (2/15) / EcoRI: expected 3 bands at 5.7, 2.1, 1.4 kb
  • Results: Lanes 1 and 3 look as expected. Lane two has one extra band at 3.5 kb corresponding to partial digestion with XbaI. 5/14 XbaI tube is ineffective and has been thrown out

July 14

Objective: insert crRNAs into pdCas9

Sequencing Order #1608

    • BigDye reaction with BD buffer 1.1
    • pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9up1
    • pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9up1
    • pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9up1
    • pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9up1
    • pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9up1
    • pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9up1
    • pdCas9-crRNA_GFP3 sample 3 w/ pdCas9up1
    • pdCas9-crRNA_GFP1 sample 7-1 w/ pdCas9dn1
    • pdCas9-crRNA_GFP1 sample 7-2 w/ pdCas9dn1
    • pdCas9-crRNA_GFP1 sample 7-3 w/ pdCas9dn1
    • pdCas9-crRNA_GFP2 sample 7-7 w/ pdCas9dn1
    • pdCas9-crRNA_GFP2 sample 7-8 w/ pdCas9dn1
    • pdCas9-crRNA_GFP2 sample 7-9 w/ pdCas9dn1
    • pdCas9-crRNA_GFP3 sample 3 w/ pdCas9dn1

Results:

    • pdCas9-crRNA_GFP1 use sample 7-1
    • pdCas9-crRNA_GFP2 use sample 7-9
    • pdCas9-crRNA_GFP3 need to find more colonies
Objective: Create pSB1C3-R0011-I13500 as destination of mCherry G-block

Prep scale digest of pSB1C3-I13500 with XbaI/PstI

  • Tube 3 from 7/11/14 miniprep
  • To extract both I13500 and pSB1C3 backbone
  • 100 uL reaction with 50 uL DNA in cutsmart

Agarose gel extraction and cleanup of pSB1C3 and I13500

  • Top band (~2kb): pSB1C3
  • Bottom band (~1kb): I13500
  • Zymoclean prep kit
  • Concentrations:
    • pSB1C3: 319 ng/uL in 20 uL (dirty)
    • I13500: 91.2 ng/uL in 20 uL (dirty)

Ligation of pSB1C3-R0011 and I13500

  • 100ng = 0.7 uL pSB1C3-R0011 (#2) cut with SpeI/PstI on 7/11
  • 150ng = 1.8 uL I13500 cut with XbaI/PstI on 7/14
  • Experimental, BO control, and IO control
  • rt for 1 hr
  • Heat shock transformation into DH5alpha and plated on LB+Cm
Objective: Switch K608012 into pSB6A1

Ligation of pSB6A1 and K608012

  • 100 ng = 0.5 uL pSB6A1 (“Yellow” sample) cut with EcoRI/SpeI on 7/9
  • 150 ng = 2.5 uL K608012 (#2B) cut with EcoRI/SpeI on 7/11
  • Experimental, BO control, and IO control
  • rt for 1 hr
  • Heat shock transformation into DH5alpha and plated on LB+Amp
Objective: Switch dCas9-tracrRNA into pSB1C3

Prep scale digests

  • dCas9-tracr PCR with XbaI/PstI (no extraction)
  • pSB1C3-K608012 (tube 3) with XbaI/PstI (to obtain pSB1C3)
  • 100 uL reaction each with 50 uL DNA in cutsmart

PCR cleanup of dCas9-tracr digest

  • Qiagen miniprep kit
  • Concentration 38.1 ng/uL in 30 uL

Agarose gel extraction and cleanup of pSB1C3

  • gel order: left K608012, right I13500
  • extracted pSB1C3, top band of K608012 (~2 kb)
  • Zymoclean kit
  • Concentration 127.9 ng/uL in 20 uL (dirty)

Ligation of pSB1C3 and dCas9-tracrRNA PCR

  • 100 ng = 3 uL dCas9-tracrRNA PCR cut with XbaI/PstI on 7/14
  • 150 ng = 0.6 uL pSB1C3 (from I13500) cut with XbaI/PstI on 7/14
  • Experimental, BO control, and IO control
  • rt for 1 hr
  • Heat shock transformation into DH5alpha and plated on LB+Cm
Objective: Create pTet-anti-tracrRNA

Prep-scale digest of pSB1C3-R0040

  • With SpeI/PstI (no extraction necessary)
  • 100 uL reaction with 50 uL DNA in cutsmart
  • 4 hrs at 37C

PCR anti-tracrRNA from pdCas9

  • Oligos dCas9tracr-up and tracrRNA-dn
  • 0, 0.4, 0.7, and 1.0 uL pdCas9 template in 50 uL reactions
  • Q5 polymerase
  • annealed at 64C, 20 sec extension

Agarose gel of anti-tracrRNA PCR

  • Results: only 1.0 uL template lane appeared to amplify
    • Need to make more using this template concentration

PCR anti-tracrRNA from pdCas9

  • Oligos dCas9tracr-up and tracrRNA-dn
  • 1.0 uL pdCas9 template in each 50 uL reaction
  • 8 tubes with Q5 polymerase
  • annealed at 64C, 20 sec extension

Agarose gel of anti-tracrRNA PCR and pSB1C3-R0040 digest

  • Lanes 1-8: anti-tracrRNA PCR
  • Lane 9: R0040 digest
  • Results:
    • PCR appears to work in all lanes (band size ~0.1 kb)
    • R0040 digest single band appears correct

PCR cleanup of anti-tracrRNA PCR and pSB1C3-R0040

  • Concentrations:
    • anti-tracrRNA: 83.3 ng/uL in 30 uL
    • pSB1C3-R0040: 25 ng/uL in 30 uL

July 15

Objective: Create pTet-anti-tracrRNA

Digest of anti-tracrRNA PCR

  • with XbaI, PstI-HF, and DpnI
  • 3 hrs at 37C
  • 60 uL DNA in 100 uL total reaction

PCR Cleanup of anti-tracrRNA PCR digest and pSB1C3-R0040 digest

  • Concentrations
    • anti-tracrRNA: 100 ng/uL
    • pSB1C3-R0040: 35 ng/uL

Ligation of anti-tracrRNA and pSB1C3-R0040

  • 100 ng = 3 uL pSB1C3-R0040 cut with SpeI/PstI
  • 15 ng = 0.15 uL anti-tracrRNA PCR cut with XbaI/PstI/DpnI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Cm
Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • Experimental: 8 colonies
  • BO Control: 4 colonies
  • IO Control: 7 colonies

Culture colonies

  • 4 copies of potential pSB1C3-dCas9-tracrRNA construct

Digest of dCas9-tracrRNA PCR and heat inactivation

  • Digest with DpnI
  • 22 uL DNA in 30 uL reaction
  • 1 hr at 37C, then 30 mins at 80C to heat-inactivate
  • assumed concentration 20 ng/uL

PCR Cleanup of pSB1C3 digest

  • 2 tubes of gel-extracted and cleaned pSB1C3 cut with XbaI and PstI
  • 11 ug original yields 309.6 ng/uL in 20 uL “superclean”

Ligation of dCas9-tracrRNA PCR and pSB1C3

  • 150 ng = 0.5 uL pSB1C3 cut with XbaI and PstI
  • 100 ng = 5 uL dCas9-tracrRNA PCR cut with Xbal/PstI/DpnI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Cm
Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block

Plate results:

  • Lawn of colonies on Experimental and BO control
  • No colonies on IO control

Digest of pSB1C3-R0011

  • With SpeI, PstI, and CIP
  • 50 uL DNA in 100 uL reaction
  • 2.5 hrs at 37C

PCR Cleanup of pSB1C3-R0011 digest and I13500 digest

  • pSB1C3-R0011 concentration 175 ng/uL
  • I13500 (from previous digestion and extraction)
    • 2.3 ug original yields 124 ng/uL in 20 uL “superclean”

Ligation of I13500 and pSB1C3-R0011

  • 100 ng = 0.6 uL pSB1C3-R0011 cut with SpeI/PstI/CIP
  • 150 ng = 1.2 uL I13500 cut with XbaI/PstI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Cm
Objective: Switch K608012 into pSB6A1

Plate results:

  • 2 colonies on experimental plate
  • 1 colony on BO, no colonies on IO

Cultured colonies

  • 2 colonies of potential pSB6A1-K608012 construct

PCR Cleanup of pSB6A1 digest and K608012 digest

  • pSB6A1, 2 tubes gel extracted and cleaned
    • 10 ug original yields 203 ng/uL in 30 uL
  • K608012, 3 tubes gel extracted and cleaned
    • 4 ug original yields 73.0 ng/uL in 30 uL

Ligation of K608012 and pSB6A1

  • 100 ng = 0.5 uL pSB6A1 cut with EcoRI and SpeI
  • 150 ng = 2 uL K608012 cut with EcoRI and SpeI
  • Experimental, Backbone only, and Insert only
  • Transformed via heat shock into DH5alphas and plated on LB+Amp

July 16

Objective: Create pTet-anti-tracrRNA

Plate results:

  • Experimental: over 1000 colonies
  • BO control had ~500 colonies, IO control had ~50 colonies

Cultured colonies

  • 4 copies of potential pSB1C3-R0040-anti-tracrRNA
Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • Experimental: 12-15 colonies
  • BO control had 3 colonies, IO control had ~30 colonies

Minipreps

  • 4 copies of potential pSB1C3-dCas9-tracrRNA construct
  • From 7/14 ligation
  • concentrations: 537.2, 45.4, 79.1, and 71.5 ng/uL
    • Only one has normal concentration, others very low

Analytical digest of pSB1C3-dCas9-tracrRNA constructs:

  • 4 copies with NheI/XbaI

Agarose gel of digests:

  • Lanes 1-2: pSB6A1-K608012 with MfeI/XbaI (expected 4.1, 0.6 kb bands)
  • Lanes 3-6: pSB1C3-dCas9-tracrRNA with NheI/XbaI (expected 5.0, 1.5 kb bands)
  • Results: gel was fuzzy and unclear
    • lanes 2,3,5,6 clearly incorrect: 1 band ~2.0 kb
    • lane 1 had one fuzzy band around 4-5 kb (could be incomplete digest)
    • lane 4 had band around 5 kb with smear above (could be incomplete digest)

New analytical digest of pSB1C3-dCas9-tracrRNA construct

  • Only copy #2 (showed potential in previous digest
  • With KpnI/SpeI for 2 hours at 37C
  • Also put previous digest back in bath for 2 more hours

Agarose gel of digests

  • DETAILS
Objective: Create pSB1C3-R0011-I13500 as destination for mCherry G-block

Plate results:

  • ~500 colonies on experimental plate
  • BO control had over 1000 colonies
  • IO control had no colonies

Cultured colonies

  • 4 copies of potential pSB1C3-R0011-I13500 construct
Objective: Switch K608012 into pSB6A1

Plate results:

  • 0 colonies on experimental plate
  • 4 colonies on BO control, 1 colony on IO control
Objective: Insert crRNAs into pdCas9

Colony PCR of pdCas9-GFP3 plate

  • Plate from 7/7/14
  • 15 colonies plus pdCas9 miniprep as control
  • double dipped in 50 uL Taq poly mix and 50 uL SOC
  • Oligos pdCas9-up1 and pdCas9-dn1
  • TALCOLONYPCR protocol with 64C anneal

Agarose gel of colony PCR

  • Expected to see higher band matching control in successful colonies
  • lanes 1 and 17 are pdCas9 control
  • assay looks helpful, successes in samples 3,5,6,8,9,12,13,15

Cultured colonies

  • Copies 3,5,6, and 8 from colony PCR in LB+Cm

July 17

Objective: Create pTet-anti-tracrRNA and pSB1C3-R0011-I13500

Minipreps

  • 4 copies of pSB1C3-R0040-anti-tracrRNA from 7/15 ligation
  • 4 copies of pSB1C3-R0011-I13500 from 7/15 ligation

Analytical digest of minipreps

  • pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI
  • pSB1C3-R0011-I13500 with MfeI/NdeI/SacI

Agarose gel of digest results:

  • Lanes 1-4: pSB1C3-R0040-anti-tracrRNA with EcoRI/PstI (expected 2.0, 0.2 kb bands) (incomplete expected 2.0, <0.1 kb bands)
  • Lanes 5-8: pSB1C3-R0011-I13500 with MfeI/NdeI/SacI (expected 1.3, 0.9, 0.33,0.29 kb bands)
  • Results:
  • R0040-antitracrRNA looks correct for all 4 copies--small band is hard to tell exactly
  • R0011-I13500 does not look correct (only one band at 2.0 kb)
Objective: Switch dCas9-tracrRNA into pSB1C3

Colony PCR on pSB1C3-dCas9-tracrRNA plate

  • Ligation plate from 7/15/14
  • 15 colonies doubled into 50 uL Taq poly mix and 50 uL SOC
  • oligos SB1C3-up1 and SB1C3-dn1
  • annealed at 65C, extension 2 mins

Agarose gel of colony PCR

  • results: primer-bands only (no successes)
Objective: Switch K608012 into pSB6A1

Culture colonies

  • 2 copies of pSB6A1-J04450 (from 6-9-14 plate)
  • 2 copies of pSB1C3-K608012

July 18

Objective: Switch dCas9-tracrRNA into pSB1C3

        PCR of dCas9-tracrRNA from pdCas9

  • 8 tubes, each with 1 uL pdCas9 template at 1 ng/uL
  • oligos dCas9tracr-up and dCas9-tracr-dn
  • 2 min extension, 64C anneal

        Agarose gel of PCR product

  • All 8 tubes look successful (band ~4 kb)

        PCR cleanup of dCas9-tracrRNA

  • Qiagen kit
  • Into 2 tubes, 30 uL each

        Prep-scale digest of PCR product

  • with PstI/XbaI/DpnI
  • in 100 uL each for 3 hrs at 37C

        Treat pSB1C3 digest with CIP

  • 1 hr at 37C

Objective: Switch K608012 into pSB6A1

        Miniprep pSB1C3-K608012 and pSB6A1-J04450

  • 1 copy each (the second copy of each did not grow in culture)
  • concentrations:
  • pSB1C3-K608012: 344 ng/uL
  • pSB6A1-J04450: 421.5 ng/uL

Prep-scale digest of K608012 and pSB6A1

  • 50 uL DNA in 100 uL reaction
  • Both with EcoRI and PstI
  • 3 hrs at 37C

        CIP treatment of pSB6A1 and heat-inactivation

  • 2.5 uL CIP added to digest tube
  • Both digests returned to 37C for 1 hr
  • Then 30 mins at 80C to heat-inactivate

        Gel extraction of pSB6A1 and K608012

  • Gel had no EtBr added, and was discarded (product was lost)

July 20

Objective: culture colonies for various purposes

        Cultured colonies

  • 4 copies pSB6A1-J04450
  • 2 from older plate, 2 from newer
  • 4 copies pSB1C3-K608012
  • 2 from older plate, 2 from newer
  • 4 copies pSB1C3-B0030
  • To obtain pSB1C3 without gel extraction
  • 4 copies pSB1C3-R0011
  • 4 copies pSB1C3-I13500

July 21

Objective: Sequence to confirm constructs

        Sequencing reaction and Order #1634

  • 1-4: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-up1
  • 5-8: pdCas9-GFP3 #3,5,6,8 with primer pdCas9-dn1
  • 9-10: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-up1
  • 11-12: pSB1C3-R0040-anti-tracrRNA #1,2 with primer SB1C3-dn1
  • Big Dye, buffer version 1.1

        Order 1634 Results:

  • pdCas9-GFP3 copies 3, 6, and 8 are correct
  • copy 5 has a single mutation
  • anti-tracrRNA: both copies are correct
  • copy 1 was a cleaner, more reliable

Objective: Create pSB1C3-R0011-I13500

        Minipreps

  • Qiagen kit
  • 4 tubes of pSB1C3-R0011
  • concentrations 255.7, 169.2, 204.8, 151.2 ng/uL
  • 4 tubes of pSB1C3-I13500
  • concentrations 148.9, 214.2, 260.7, 432.5

Prep scale digests of pSB1C3-R0011 and I13500

  • pSB1C3-R0011 with SpeI/PstI, samples 1,3
  • pSB1C3-I13500 with XbaI/PstI, samples 1,3
  • 50 uL DNA in 100 uL each reaction, 2 tubes per construct
  • 3 hrs at 37C
  • Added CIP (2.5 uL) to pSB1C3-R0011 reaction
  • 1 more hr for both at 37C
  • Heat inactivation of both reactions: 30 mins at 80C

        Gel extraction of I13500

  • extraction gel 1, lanes 1 and 2
  • Zymoclean prep kit
  • sample 1 (lane 1): 300 mg gel yields
  • sample 3 (lane 2): 550 mg gel yields

        PCR cleanup of pSB1C3-R0011

  • Qiagen kit
  • Two tubes of digest combined into one tube product
  • Concentration

        Ligation of pSB1C3-R0011 and I13500

  • Old backbone (pSB1C3-R0011 digested 7/15): 100 ng = 0.6 uL
  • New backbone (pSB1C3-R0011 digested 7/21): 100 ng = 2.2 uL
  • Old insert (I13500 extracted 7/15): 300 ng = 2.4 uL
  • New insert (I13500 extracted 7/21): 300 ng = 3 uL
  • Ligations numbered 1-8:
  • 1. OB+OI
  • 2. OB+NI
  • 3. NB+OI
  • 4. NB+NI
  • 5. OB control
  • 6. OI control
  • 7. NB control
  • 8. NI control
  • 1 hr at rt
  • Transformed via heat shock into DH5alphas, plated on LB+Cm

Objective: Switch dCas9-tracrRNA into pSB1C3

        Minipreps

  • Qiagen kit
  • 4 tubes of pSB1C3-B0030
  • to obtain pSB1C3 without gel extraction
  • concentrations 116.8, 99.9, 115.3, 145.1 ng/uL

PCR cleanup of dCas9-tracrRNA PCR

  • Product digested 7/18 with XbaI/PstI/DpnI
  • Concentration 80.5 ng/uL in 30 uL
  • Heat inactivation of cleaned product, 30 mins at 80C

Prep scale digest of pSB1C3-B0030

  • With XbaI/PstI, samples 1,3
  • 50 uL DNA in 100 uL each reaction, 2 tubes
  • 3 hrs at 37C
  • Added CIP (2.5 uL)
  • 1 more hr for both at 37C
  • Heat inactivation: 30 mins at 80C

        PCR cleanup of pSB1C3

  • B0030 should not be retained
  • Qiagen kit
  • Two tubes of digest combined into one tube product
  • Concentration

        Ligation of pSB1C3 and dCas9-tracrRNA

  • Backbone (pSB1C3 digested 7/21): 300 ng = 3.5 uL
  • Old insert (dCas9-tracrRNA digested 7/15): 100 ng = 3 uL
  • New insert (dCas9-tracrRNA digested 7/18): 100 ng = 1.2 uL
  • Ligations numbered 9-13:
  • 9. B+OI
  • 10. B+NI
  • 11. B control
  • 12. OI control
  • 13. NI control
  • 1 hr at rt
  • Transformed via heat shock into DH5alphas, plated on LB+Cm

Objective: Switch K608012 into pSB6A1

        Minipreps

  • Qiagen kit
  • 4 tubes of pSB6A1-J04450
  • concentrations 189.3, 432.5, 688.7, 487.3 ng/uL
  • 4 tubes of pSB1C3-K608012
  • concentrations 68.8, 91.6, 346.1, ng/uL

Prep scale digests of pSB6A1-J04450 and pSB1C3-K608012

  • pSB6A1-J04450 with XbaI/SpeI, samples 1,3
  • pSB1C3-K608012 with EcoRI/PstI, samples 1,3
  • 50 uL DNA in 100 uL each reaction, 2 tubes per construct
  • 3 hrs at 37C
  • Added CIP (2.5 uL) to pSB6A1-J04450 reaction
  • 1 more hr for both at 37C
  • Heat inactivation of both reactions: 30 mins at 80C

        Gel extraction of pSB6A1 and K608012

  • K608012: gel 1, lane 3 and gel 2, lane 1
  • pSB6A1: gel 2, lanes 2 and 3
  • Zymoclean prep kit
  • K608012 sample 1 (lane 1-3): 300 mg gel yields
  • K608012 sample 3 (lane 2-1): 550 mg gel yields
  • pSB6A1 sample 1 (lane 2-2):
  • pSB6A1 sample 3 (lane 2-3):

        Ligation of pSB6A1 and K608012

  • Old backbone (pSB6A1 extracted 7/15): 100 ng = 0.5 uL
  • Old insert (K608012 extracted 7/15): 300 ng = 4 uL
  • Ligations numbered 14-16:
  • 14. B+I
  • 15. B control
  • 16. I control
  • 1 hr at rt
  • Transformed via heat shock into DH5alphas, plated on LB+Amp

        Gibson of pSB6A1 and K608012

  • New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
  • New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
  • Numbered 17-19:
  • 17. B+I
  • 18. B control
  • 19. I control
  • 15 uL Mert’s DIY Gibson mix
  • 1 hr at 60C
  • Transformed via heat shock into DH5alphas, plated on LB+Amp

SLIC of pSB6A1 and K608012

  • New backbone (pSB6A1 extracted 7/21): 100 ng = 0.3 uL
  • New insert (K608012 extracted 7/21): 300 ng = 1.7 uL
  • Numbered 20-22:
  • 20. B+I
  • 21. B control
  • 22. I control
  • 2.5 mins at rt, then 10 mins at 4C
  • Transformed via heat shock into DH5alphas, plated on LB+Amp

July 22

Objective: Create pSB1C3-R0011-I13500

        Plate results:

  • All four experimental plates had lots of colonies
  • both Backbone-only controls had similar colony numbers to experimentals
  • Insert-only controls had no colonies

        Colony PCR

  • 4 copies each of 4 experimental plates = 16 colonies
  • Oligos SB1C3-up and SB1C3-dn with Taq polymerase
  • 66C anneal temp, 2 min extension time

        Agarose gel of colony PCR results

  • 1-4: OB+OI pSB1C3-R0011-I13500
  • 5-8: OB+NI pSB1C3-R0011-I13500
  • 9-12: NB+OI pSB1C3-R0011-I13500
  • 13-16: NB+NI pSB1C3-R0011-I13500
  • 17-20: B+OI pSB1C3-dCas9-tracrRNA
  • 21-24: B+NI pSB1C3-dCas9-tracrRNA
  • Results: no successful bands on any colonies
  • Just primer-dimers or small amplified bands?

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • Both experimental plates had lots of colonies
  • Backbone-only controls had similar colony numbers to experimentals
  • Insert-only controls had no colonies

        Colony PCR

  • 4 copies each of 2 experimental plates = 8 colonies
  • Oligos SB1C3-up and SB1C3-dn with Taq polymerase
  • 66C anneal temp, 2 min extension time

        Agarose gel of colony PCR results

  • see above for gel order
  • Results: no successful bands on any colonies
  • Just primer-dimers or small amplified bands?

Objective: Switch K608012 into pSB6A1

        Plate results:

  • No colonies on Gibson or SLIC plates
  • 3 colonies on Ligation plate
  • A few colonies on controls, but not many

        Cultured colonies

  • 3 colonies of potential pSB6A1-K608012 from Ligation plate
  • in LB+Amp

Ligation of K608012 and pSB6A1

  • Same constructs as 7/21 ligation
  • This time let recover in SOC for 2 hrs instead of 1 hr
  • Plated on LB+Amp

Objective: Try putting K608012 into pSB1A2 instead

        Transformation from kit plate

  • Plate 4-1N
  • pSB1A2-B0034
  • Plated on LB+Amp

July 23

Objective: Switch K608012 into pSB6A1

        Miniprep 3 copies of potential pSB6A1-K608012

  • Qiagen kit
  • Saved stocks in 15% glycerol

        Analytical digest of minipreps

  • With NdeI in Cutsmart, 10 uL final volume

        Agarose gel of analytical digest

  • Results:
  • Copies 1 and 2 are strange and incorrect
  • Copy 3 looks correct
  • Need to check with more digests

        Analytical digest to check promising sample

  • Copy 3 in 3 different reactions
  • EcoRI/SpeI
  • EcoRI/MfeI
  • NcoI/PvuI
  • Control: pSB6A1-J04450 with EcoRI/SpeI/PvuI
  • Control: pSB1C3-K608012 with MfeI/NcoI

        Agarose gel of analytical digest

  • 1. Ligation 3 with EcoRI/SpeI
  • 2. Ligation 3 with EcoRI/MfeI
  • 3. Ligation 3 with NcoI/PvuI
  • 4. pSB6A1-J04450 with EcoRI/SpeI/PvuI
  • 5. pSB1C3-K608012 with MfeI/NcoI
  • Results:
  • Sample looks good
  • Incorrect samples 1 and 2 glycerol stocks thrown out

Objective: Switch R0040-anti-tracrRNA into pSB4K5

        Treat pSB4K5 with CIP

  • 1.25 uL CIP in 25 uL reaction
  • pSB4K5 was cut with EcoRI/SpeI and extracted on 7/9

        Prep-scale digest to obtain R0040-anti-tracrRNA

  • pSB1C3-R0040-anti-tracrRNA
  • with EcoRI/SpeI

        Gel extraction and cleanup of R0040-anti-tracrRNA

  • Extracted smaller band
  • Attempted to clean up using Qiagen prep kit
  • dissolved with Zymoclean ADB
  • Then followed Qiagen PCR cleanup protocol
  • 510 mg gel yielded little or no DNA

Objective: Create pSB1C3-R0011-I13500

        Colony PCR

  • 8 colonies from 7/21 ligation plates
  • Control colonies from plates of pSB1C3-I13500 and pSB1C3-R0011

        Agarose gel of colony PCR results

  • No successes
  • Controls did not work properly either
  • colony PCR does not seem to be effective--need to just pick colonies

        Culture colonies

  • 8 colonies of potential pSB1C3-R0011-I13500
  • From 7/21 ligation plates (colonies from today’s colony PCR)

Objective: Measurement interlab study

        Transformations from kit plates

  • Plate 1-20K
  • pSB1C3-K823005
  • Plate 1-22I
  • pSB1C3-K823012
  • Plate 2-24B
  • pSB1C3-E0240

July 24

Objective: Create pSB1C3-R0011-I13500

Miniprep 8 colonies of potential pSB1C3-R0011-I13500

  • Qiagen kit

Analytical digest of minipreps

  • 8 copies with NcoI
  • 8 copies with EcoRI/SpeI

Agarose gel of digest results

  • 1-8: copies 1-8 with NcoI
  • 9-16: copies 1-8 with EcoRI/SpeI
  • Results: no successes, insert appears to be missing
  • Unsure whether incorrect colonies are pSB1C3 or pSB1C3-R0011

Agarose gel of digest results

  • Thicker gel, longer run time, more DNA
  • from same digest tubes
  • Results:
  • no small band on EcoRI/SpeI cut and no difference in larger band for the two cuts, indicating that R0011 is not present

Ligation of pSB1C3-R0011 and I13500

  • Used lab stock of T4 Ligase and buffer rather than iGEM stock
  • 100 ng pSB1C3-R0011 = 2.5 uL
  • 300 ng I13500 = 3 uL
  • 4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
  • BO and IO controls for each time
  • Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)

Objective: Switch dCas9-tracrRNA into pSB1C3

Ligation of pSB1C3 and dCas9-tracrRNA

  • Used lab stock of T4 Ligase and buffer rather than iGEM stock
  • 300 ng pSB1C3 = 3 uL
  • 100 ng dCas9-tracrRNA PCR = 1 uL
  • 4 tubes of ligation, run at rt with Ligase for 15, 30, 45, and 60 mins
  • BO and IO controls for each time
  • Plated on LB+Cm, two tubes per plate (used wooden streaker rods to spread on half)

Objective: Test GFP repression by crRNAs

Transform pSB6A1-K608012 into DH5alpha-ZI

  • Plated on LB+Amp

Objective: Obtain constructs for various uses

Streaked plate from frozen stock

  • One LB+Cm plate in thirds:
  • pSB1C3-R0040-antitracrRNA
  • pSB1C3-Repeat-seq scaffold
  • pSB1C3-Csy4-gRNA scaffold

Objective: Measurement interlab study

Cultured colonies (2 each)

  • pSB1C3-E0240
  • pSB1C3-K823005
  • pSB1C3-K823012
  • pSB3K3-I20260

July 25

Objective: Create pSB1C3-R0011-I13500

Plate results:

  • no apparent difference between 15, 30, 45, or 60 minute ligations
  • many colonies on all Experimental and BO control plates
  • no colonies on IO control

Colony PCR:

  • 16 colonies from pSB1C3-R0011-I13500 plates
  • 4 from each ligation time
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • 66C anneal temp, 45 sec extension

Agarose gel of colony PCR

  • No successes
  • It seems as though colony PCRs are not working--try control with a gradient

Gradient Colony PCR

  • to optimize with SB1C3-up and SB1C3-dn primers
  • template colonies of pSB1C3-K608011 from plate
  • anneal temps 62-72 C, extension time 45 sec

Agarose gel of gradient colony PCR

  • left to right: 62C to 72C anneal temps
  • Results: 62C was the best anneal temp--worked well
  • gradual decrease as temp increased, no amplification above 65C

Objective: Switch dCas9-tracrRNA into pSB1C3

Plate results:

  • no apparent difference between 15, 30, 45, or 60 minute ligations
  • many colonies on all Experimental and BO control plates
  • no colonies on IO control

Colony PCR:

  • 16 colonies from pSB1C3-dCas9-tracrRNA plates
  • 4 from each ligation time
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • 66C anneal temp, 45 sec extension

Agarose gel of colony PCR

  • No successes
  • It seems as though colony PCRs are not working--try control with a gradient
  • see gradient results above

Objective: Various ligations

Prep-scale digests

  • pSB1C3-Csy4-scaffold (2 copies) with XbaI/PstI
  • to ligate into R0040
  • pSB1C3-Repeat-seq scaffold (2 copies) with XbaI/PstI
  • to ligate into R0040
  • pSB1C3-R0040-anti-tracrRNA (2 copies) with XbaI/PstI
  • 100 uL reactions each tube

PCR cleanups on all digests

  • Qiagen kit
  • Concentrations (in 50 uL each):
  • pSB1C3-Csy4-scaffold: 55.9, 105.5 ng/uL
  • pSB1C3-Repeat-seq scaffold: 15.2, 55.2 ng/uL
  • pSB1C3-R0040-anti-tracrRNA: 90.6 ng/uL
  • Need to extract from gels before using, only cleaned up for safer storage

Objective: Measurement interlab study

Minipreps

  • Qiagen kit
  • pSB1C3-K823005 (2 copies)
  • concentrations 256.3, 314.8 ng/uL
  • pSB1C3-K823012 (2 copies)
  • concentrations 204.4, 192.5 ng/uL
  • pSB1C3-E0240 (2 copies)
  • concentrations 266.3, 241.5 ng/uL

Prep-scale digests

  • pSB1C3-K823005 (2 copies) with SpeI/PstI
  • pSB1C3-K823012 (2 copies) with SpeI/PstI
  • pSB1C3-E0240 (2 copies) with XbaI/PstI
  • 100 uL each tube

PCR cleanups on all digests

  • Qiagen kit
  • Concentrations (in 50 uL each):
  • pSB1C3-K823005: 156.3, 181.7 ng/uL
  • pSB1C3-K823012: 110.0, 98.0 ng/uL
  • pSB1C3-E0240: 110.0, 114.6 ng/uL

Objective: Test crRNA repression of GFP reporter

Cultured colony

  • To make CCEC of reporter strain
  • One colony of pSB6A1-K608012 in DH5alpha-ZI
  • Grew for 8 hrs during the day in 2 mL LB+Amp at 37C
  • Inoculated into 100 mL flask of LB+Amp for overnight shaker at rt

Flow cytometry of reporter strain

  • quick check to make sure reporter still works with new backbone
  • Used parameters from previous GFP flow data
  • Levels slightly under what was seen before, but still strong

July 26

Objective: Test crRNA repression of GFP reporter

Prepared Chemically competent E. coli

  • Using DH5alpha-ZI + pSB6A1-K608012
  • iGEM CCEC protocol with smaller volumes (made ~20 1-mL aliquots)

Double-Transformation of pdCas9 variants into reporter cells

Charlie Cooper

  • pdCas9 (with BsaI insert as crRNA sequence)
  • pdCas9-crRNA_GFP1
  • pdCas9-crRNA_GFP2
  • pdCas9-crRNA_GFP3

Objective: Create pSB1C3-R0011-I13500

Colony PCRs

  • 16 colonies from 7/24 ligation plates
  • oligos SB1C3-up and SB1C3-dn
  • 62C anneal (as determined by gradient), 45 sec extension

Agarose gel of Colony PCRs

  • Results: colonies 5 and 14 amplified at the correct size

Culture colonies

  • Colonies 5 and 14 from colony PCR of potential pSB1C3-R0011-I13500

Objective: Create pSB1C3-dCas9-tracrRNA

Colony PCRs

  • 16 colonies from 7/24 ligation plates
  • oligos SB1C3-up and SB1C3-dn
  • 62C anneal (as determined by gradient), 45 sec extension
  • PCRs left in machine without starting reaction for ~1 hr, could have kill reaction

Agarose gel of Colony PCRs

  • No successes

July 27

Objective: Test crRNA repression of GFP reporter

Cultured colonies for flow

  • 3 copies of each
  • DH5alpha-Z1 (background)
  • DH5-alpha-Z1 + pSB6A1-K608012 (reporter positive control)
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9 (non-target control)
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP1
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP2
  • DH5-alpha-Z1 + pSB6A1-K608012 + pdCas9-crRNA_GFP3

Objective: Create pSB1C3-R0011-I13500

Minipreps

  • Colonies 5 and 14 from 7/26 colony PCRs
  • Qiagen kit

July 28

Objective: Create pSB1C3-R0011-I13500

        Analytical digest of minipreps

  • Colonies 5 and 14 of potential pSB1C3-R0011-I13500
  • with MfeI
  • with NcoI
  • with EcoRI/PstI

        Agarose gel of analytical digest results

  • Gel Order:
  • #14 w/ MfeI
  • #14 w/ EcoRI/PstI
  • #14 w/ NcoI
  • #5 w/ MfeI
  • #5 w/ EcoRI/PstI
  • #5 w/ NcoI
  • Results: All looks correct, except that MfeI cut once instead of twice
  • could be pSB1C3-I13500 without R0011

Objective: Insert mCherry G-block into pSB1C3-R0011-I13500

        PCR of mCherry G-block

  • Q5 polymerase with Oligos SB1C3-dn and pDGC3-up
  • 4 tubes with 0, 0.4, 0.7, 1.0 uL template at 1 ng/uL
  • 45 sec extension, 66C anneal temp

        Agarose gel of PCR

  • 3 tubes look successful

        PCR cleanup of mCherry G-block PCR

  • Qiagen kit
  • 40 uL at 27.6 ng/uL

Objective: Various ligations

        Gel extraction and cleanup

  • Each extraction included 2 cleaned digests combined into one lane (~100 uL)
  • Cut and cleaned with Zymoclean prep kit
  • pSB1C3-Csy4-scaffold cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 47.5 ng/uL in 30 uL
  • Csy4-scaffold insert 15.4 ng/uL in 30 uL
  • pSB1C3-Repeat-seq scaffold cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 74.8 ng/uL in 30 uL
  • Repeat-seq scaffold insert 13.1 ng/uL in 30 uL
  • pSB1C3-R0040-anti-tracrRNA cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 164.0 ng/uL in 30 uL
  • R0040-anti-tracrRNA 176.0 ng/uL in 30 uL

PCR Cleanup on gel extraction cleanups

  • to “superclean”
  • pSB1C3 4 tubes combined into 1: 469.6 ng/uL in 20 uL
  • Repeat-seq scaffold: 23.9 ng/uL
  • rough graph, probably not useful
  • Csy4 scaffold: 16 ng/uL
  • rough graph, probably not useful
  • R0040-anti-tracrRNA: 7.4 ng/uL
  • rough graph, probably not useful

Objective: Measurement interlab study

        CIP treatment of backbones

  • Using Charlie’s CIP stock
  • pSB1C3-K823005 cut with SpeI/PstI on 7/25 (2 tubes)
  • pSB1C3-K823012 cut with SpeI/PstI on 7/25 (2 tubes)

        Gel extraction and cleanup

  • 2 cleaned digests combined into one lane (~100 uL)
  • Cut and cleaned with Zymoclean prep kit
  • pSB1C3-E0240 cut with XbaI/PstI on 7/25
  • pSB1C3 backbone 116.4 ng/uL in 30 uL
  • E0240 86.5 ng/uL in 30 uL

        PCR Cleanup of CIP treatment and gel extraction cleanup

  • Qiagen kit
  • pSB1C3-K823005 SpeI/PstI CIP treated: 119.8, 119.7 ng/uL
  • pSB1C3-K823012 SpeI/PstI CIP treated: 60.9, 62.3 ng/uL
  • E0240 XbaI/PstI extracted: 56.5 ng/uL in 20 uL (“superclean”)

        Ligations

  • E0240 into two backbones: pSB1C3-K823005 and pSB1C3-K823012
  • pSB1C3-K823005 SpeI/PstI/CIP: 100 ng = 0.87 uL
  • pSB1C3-K823012 SpeI/PstI/CIP: 100 ng = 1.67 uL
  • E0240 XbaI/PstI: 300 ng = 6 uL
  • 2 BO controls and 1 IO control
  • 20 mins at rt
  • Heat shock transformation and plated on LB+Cm

Objective: Test crRNA repression of GFP reporter

        Dilute cultures to grow in log phase for flow

  • 1/1000 dilution into 50 mL
  • 18 samples: 3 copies each of 6 strains (labelled 2-19)
  • Samples 2-4: DH5alpha-Z1 (background)
  • grown in LB+Spec
  • Samples 5-7: DH5alpha-Z1 + pSB6A1-K608012 (reporter positive control)
  • grown in LB+Amp
  • Samples 8-10: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9 (non-target control)
  • grown in LB+Amp+Cm
  • Samples 11-13: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP1
  • grown in LB+Amp+Cm
  • Samples 14-16: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP2
  • grown in LB+Amp+Cm
  • Samples 17-19: DH5alpha-Z1 + pSB6A1-K608012 +pdCas9_GFP3
  • grown in LB+Amp+Cm

Flow cytometry

  • Samples grown for 4.5 hrs after initial dilution
  • 1/50 final dilution of sample into PBS, placed on ice until run
  • parameters (same as previous GFP runs):
  • FSC 350V log3
  • SSC 350V log3
  • B1 350V log5
  • Trigger 10
  • 10,000 event limit (100,000 events used in the past)
  • Results
  • ~3-fold repression of reporter with crRNA_GFP1 or crRNA_GFP3
  • crRNA_GFP2 did not repress
  • non-target control repressed little or not at all
  • See document “Flow stats 7-28-14” for data details

July 29

Objective: Switch R0040-anti-tracrRNA into pSB4K5

        Minipreps

  • pSB4K5-J04450 (4 copies)
  • pSB1C3-R0040-anti-tracrRNA (4 copies)

        Prep-scale digest

  • pSB4K5-J04450 (4 copies) with EcoRI/SpeI
  • pSB1C3-R0040-anti-tracrRNA (4 copies) with EcoRI/SpeI

        Gel extraction and cleanup of digests

  • Cut pSB4K5 backbone from 4 copies of pSB4K5-J04450
  • 2 digests per gel well
  • 2.04 g total gel eluted into two tubes:
  • 51.4 ng/uL and 94.5 ng/uL, 30 uL each
  • Cut R0040-anti-tracrRNA insert from pSB1C3-R0040-anti-tracrRNA
  • 2 digests per gel well
  • 1.61 g total gel eluted into two tubes:
  • 99.4 ng/uL and 82.3 ng/uL, 30 uL each

Objective: Create pSB1C3-R0011-I13500

        Colony PCR

  • 24 colonies of potential pSB1C3-R0011-I13500
  • 8 colonies from 7/15 ligation
  • 4x4=16 colonies from 4 different 7/21 ligation plates
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • extension time 45 sec, anneal temp 62C

Agarose gel of colony PCR results

  • colony #19 is only promising result

        Analytical digest

  • pSB1C3-R0011 copies 2, 4
  • pSB1C3-I13500 copies 2, 4
  • with MfeI/NcoI
  • To test whether enzymes or original parts are the source of ligation difficulties

        Agarose gel of digest results

  • Order:
  • 1. pSB1C3-R0011 #2 cut with MfeI/NcoI (expected 2 bands)
  • 2. pSB1C3-R0011 #4 cut with MfeI/NcoI (expected 2 bands)
  • 3. pSB1C3-I13500 #2 cut with MfeI/NcoI (expected 3 bands)
  • 4. pSB1C3-I13500 #4 cut with MfeI/NcoI (expected 3 bands)
  • Results:
  • R0011 both copies only show one band
  • I13500 both copies appear correct

        Cultured colonies

  • Potential pSB1C3-R0011-I13500 #19 from colony PCR
  • pSB1C3-R0011 (3 copies) from original transformation
  • suspicion that our R0011 is incorrect--could be just bad copies

Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds

        Culture colonies

  • pSB1C3-R0040 (4 copies)

Objective: Measurement interlab study

        Plate results

  • both experimental and both backbone-only plates all had hundreds of colonies
  • insert-only controls had no colonies

        Colony PCR

  • 8 colonies of potential pSB1C3-K823005-E0240
  • 8 colonies of potential pSB1C3-K823012-E0240
  • Taq polymerase with oligos SB1C3-up and SB1C3-dn
  • extension time 45 sec, anneal temp 62C

        Agarose gel of colony PCR results

  • 1-8: pSB1C3-K823005-E0240
  • 9-16: pSB1C3-K823012-E0240
  • Results: no successes

        

July 30

Objective: Create pSB1C3-R0011-I13500

        Minipreps

  • pSB1C3-R0011-I13500 #19
  • concentration 246.4 ng/uL
  • pSB1C3-R0011 (3 copies)
  • concentrations 187.6, 178.8, 212.8 ng/uL

Analytical digest

  • pSB1C3-R0011-I13500 #19
  • three digests in cutsmart, 10 uL final volume each
  • NcoI
  • MfeI
  • XbaI/PstI
  • pSB1C3-R0011 (3 copies)
  • with NcoI/MfeI in cutsmart, 10 uL final volume

Agarose gel of digest results

  • 1. pSB1C3-R0011-I13500 #19 with NcoI
  • 2. pSB1C3-R0011-I13500 #19 with MfeI
  • 3. pSB1C3-R0011-I13500 #19 with EcoRI/PstI
  • 4. pSB1C3-R0011 #1 with NcoI/MfeI
  • 5. pSB1C3-R0011 #2 with NcoI/MfeI
  • 6. pSB1C3-R0011 #3 with NcoI/MfeI
  • Results: hard to explain
  • R0011-I13500 not correct
  • R0011 all three copies only cut once

Objective: Switch R0040-anti-tracrRNA into pSB4K5

        Treat pSB4K5 with Antarctic Phosphatase

  • 2 copies digested on 7/29, combined into one tube with 100 uL final volume
  • 2 hrs at 37C

        PCR cleanup of AP-treated pSB4K5

  • Qiagen kit
  • concentration 45.2 ng/uL in 50 uL

        Ligation of pSB4K5 and R0040-anti-tracrRNA

  • 2 experimental plates with 3x and 6x molar insert
  • pSB4K5 backbone (EcoRI/SpeI/AP)100 ng = 2 uL
  • R0040-anti-tracrRNA insert (EcoRI/SpeI) 30 ng = 0.3 uL, or 60 ng = 0.6 uL
  • Backbone-only and insert-only controls
  • Transformed via heat shock and plated on LB+G418

Objective: Put R0040 promoter in front of crRNA and gRNA scaffolds

        Minipreps

  • pSB1C3-R0040 (4 copies)
  • concentrations 47.4, 80.7, 109.7, 193.5

Objective: Switch dCas9-tracrRNA into pSB1C3

        Treat pSB1C3 with Antarctic Phosphatase

  • cleaned copy from 7/28 with 100 uL final volume
  • 2 hrs at 37C

        PCR cleanup of AP-treated pSB1C3

  • Qiagen kit
  • concentration 104.5 ng/uL in 50 uL

        Ligation of pSB1C3 and dCas9-tracrRNA

  • 2 experimental plates
  • 3x molar dCas9-tracrRNA “insert”
  • pSB1C3 100 ng = 1 uL
  • dCas9-tracrRNA 600 ng = 7.5 uL
  • 3x molar pSB1C3 “backbone”
  • pSB1C3 150 ng = 1.5 uL
  • dCas9-tracrRNA 100 ng = 1.2 uL
  • Backbone-only and insert-only controls
  • Transformed via heat shock and plated on LB+Cm

July 31

Objective: Switch R0040-anti-tracrRNA into pSB4K5

Plate results:

  • Hundreds of colonies on both experimental plates
  • 20-30 colonies on backbone-only control
  • 0 colonies on insert-only control

Cultured colonies

  • 4 copies of pSB4K5-R0040-anti-tracrRNA

Objective: Switch dCas9-tracrRNA and dCas9-tracrRNA-crRNA into pSB1C3

Plate results:

  • Experimental plate 2 (higher pSB1C3 concentration) had 2 colonies
  • Experimental plate 1 had no colonies
  • Backbone-only control had 1 colony, insert-only control had 0

Cultured colonies

  • 2 copies of potential pSB1C3-dCas9-tracrRNA

PCRs of tracrRNA-dCas9 and tracrRNA-dCas9-crRNA

  • 8 tubes of each, with 1 uL 1 ng/uL pdCas9 template per tube
  • tracrRNA-dCas9 oligos: dCas9tracr-up and dCas9tracr-dn
  • tracrRNA-dCas9-crRNA oligos: dCas9tracr-up and pdCas9-dn
  • Q5 polymerase
  • 64C anneal temp, 2:30 extension time

Agarose gel of PCRs

  • 1-8: tracrRNA-dCas9-crRNA
  • 9-16: tracrRNA-dCas9
  • Results: all 16 lanes look good

PCR cleanup of PCRs

  • 8 tubes into 50 uL for each of two PCRs
  • Concentrations
  • tracrRNA-dCas9-crRNA: 350 ng/uL
  • tracrRNA-dCas9: 335 ng/uL

Prep-scale digest of 2 PCRs

  • tracrRNA-dCas9-crRNA and tracrRNA-dCas9
  • Each in 100 uL final volume with 2 uL each of XbaI/PstI/DpnI

PCR cleanup of digests

  • Into 50 uL final volume

Objective: Put R0040 in front of Csy4 scaffold and Repeat-seq scaffold

Prep-scale digest

  • pSB1C3-R0040 (2 copies) with 2.5 uL each of SpeI/PstI
  • 100 uL final volume each copy

PCR cleanup of digests

  • Into 50 uL final volume

Objective: Triple-transformation of R0040-anti-tracrRNA into Z1-reporter-crRNA_GFP cells

Cultured cells in morning

  • straight from frozen stocks into 2 mL LB+Cm+Amp
  • DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP1
  • DH5alpha-ZI + pSB6A1-K608012 + pdCas9_GFP3

Cultured cells in evening

  • 1 mL from morning culture into 50 mL flask for overnight growth
  • To make chemically competent cells

Objective: Create pSB1C3-R0011-I13500

Cultured colonies

  • 2 copies of pSB1C3-R0011 from streak plate of original frozen stock