Team:Duke/Notebook/July

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

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

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

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

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

Objective: Test effectiveness of DH5alpha-Z1 strain

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

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

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

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)

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

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

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

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.

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

Culture pSB1C3-I13521 (pTet-RFP)

• For flow
• In aTc and non-aTc

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

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)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Plate results:

• 0 colonies on experimental plate
• 4 colonies on BO control, 1 colony on IO control

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

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)

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)

Culture colonies

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

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)

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

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

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

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

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

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

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

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

Objective: Create pSB1C3-R0011-I13500

Analytical digest of minipreps

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• 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

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

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

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