Team:Duke/Notebook/MayJun

Followed Charlie’s Cloning Protocols to prepare chemically competent E. Coli

• Once E. Coli were prepared, the solution was aliquoted into 12 labeled tubes (450l in each tube) and tubes were stored in iGEM box in -80C cooler on far left

Next Steps:

• Transformation

Transformation of CCEC with RFP Construct of varying concentrations+control with no DNA

• Followed Charlie’s Cloning Protocols
• Mistakenly used all of DNA construct from the kit… So not sure if the transformation will be successful
• Plated the transformed DNA and put in incubator at 37C
• Results (6/2/14)--Transformation unsuccessful because improper procedure was done by MFaw

Next Steps:

• Look at plates, compare cultures

Due to my failure to correctly conduct the transformation yesterday, it was necessary to redo the transformation.

• Followed Charlie’s Cloning Protocols
• Added 1 ul of .5, 5, 10, 20, 50, and 0 (control) pg/lof RFP Construct to chemically competent E. Coli (CCEC), and followed procedure
• Plated the transformed DNA on 6 separate plates, put in 37C overnight
• Results: (6/3/14)-Transformation unsuccessful. Try the transformation again with Charlie’s cells and with new cells grown using iGEM’s protocol

Next Steps:

• Look at plates, compare cultures, etc.

Prepare SOB Medium for bacterial transformation

• Protocol from iGEM’s parts website http://parts.igem.org/Help:Protocols/Competent_Cells
• Made 1 L, autoclaved and stored in two 500 mL containers in cold room
• medium still appeared cloudy before autoclave--may just be new recipe

Prepare CCMB 80 Buffer for making chemically competent E. coli cells

• Protocol from iGEM’s parts website
• Made 1 L, filtered and stored in two 500 mL containers in cold room
• pH 6.34 (overshot a few times pH adjustment, but no noticeable precipitate formed

Autoclave two 500 mL culture flasks

• For CCEC protocol
• With water inside to remove detergent residues

Next steps:

• Prepare CCEC

The second (and more properly conducted) attempt to transform the CCEC with RFP construct BBa_J04450 from 6/2 failed. Today, we are trying to see if we can get any transformed cells to grow in plates.

• Followed Charlie’s Cloning protocols,with slight modifications
• Added 5 lof 0 (control) and 50pg/lRFP Construct BBa_J04450 to Charlie’s chemically competent E. Coli, and followed procedure
• Plated the transformed DNA on 2 separate plates, put in put in 37C overnight
• Results: No colonies grew (6/5/14)

Next Steps:

• Examine plates to see if any cultures grew
• Attempt the transformation with the cell cultures growns using iGEM’s standard protocols that can be found here:
• Lab currently in the process of making these CCEC

Prepare pdCas9 and pCsy4 to be miniprepped tomorrow

• Put pdCas9 into a culture tube with 5ml SOC+Chloramphenicol
• Put pCsy4 in a culture tube with 5ml SOC+Amp
• Left both to shake at 37C to grow the plasmid DNA--Charlie will retrieve them

Next steps:

• Miniprep plasmid DNA

Results: Plates transformed 6/4 had no colonies (see for results)

Stored glycerol stocks (15% glycerol) of pdCas9 (A1) and pdCas9 (A2) at -80

Miniprep our pdCas9 and pCsy4

• We miniprepped both pdCas9 and pCsy4 following standard protocol included in the miniprep kit. We were unsure of how successful our miniprep of pdCas9 would be because it was low copy.
• We took our own miniprep kit, prepared all solutions necessary, and labeled as ours. Use this miniprep kit from now on.
• We analyzed the concentrations of our plasmids with the spectrophotometer.
• Results (6/5/14): Both minipreps were successful, with pdCas9 at concentration 103.4 ng/ulpCsy4 at concentration 138.1ng/ul
• Stored in iGEM 2014 Box 1, A1 and A2

Next steps:

• Wait for oligos ordered 6/4/14 in order to use these plasmids in construction.

Results: the colonies that we left to grow in 2mL of SOB medium in the shaker at 37C didn’t grow…. Not sure if there’s a problem with the SOB medium, the cells, etc. Today we’ll try growing various bacteria in the SOB medium to try to figure out what went wrong

PCR

• Followed standard PCR procedure on the pdCas9
• 0.5 uL Phusion HF polymerase/reaction
• 0.25 uL each oligo
• 1 uL dNTP mix
• Oligo 5: 5p-EcoR1-Xbal-Rpt
• Oligo 6: Spe1-Repeat-3p

Note: Created new box called iGEM Cloning Project. dNTP’s in A1, and EcoR1-Xbal-Rptg-Rpt-Spe1-PCR in A2

PCR was successful, with concentration 115.4 ng/ul. Pooled 3x50uL reactions with PCR cleanup protocol and eluted in to 40uL dH2O

Next steps:

• Digest and transformation for biobrick submission

Miniprepped and stored glycerol stocks of two copies each of pSB6A1-BBa_J04450 and pSB1C3-BBa_J04450

• Glycerol stocks in iGEM bacteria box 1, A3 and A4.
• Followed Qiagen kit for miniprep procedure
• All four E. coli strains visibly reddish before miniprep (sign that RFP is expressed)
• Results: All four successful, concentrations (ng/uL):
• 6A1-1: 429.2, 6A1-2: 461.2,1C3-1: 337.2, 1C3-2: 294.2

• 10 lanes:
• 1. BBa_J04450+pSB6A1-1(uncut)
• 2. BBa_J04450+pSB1C3-1 (uncut)
• 3. A-1 cut with Kpn1-expected 4.8 and 0.25 kb bands
• 4. A-2 cut with Kpn1-expected 4.8 and 0.25 kb bands
• 5. A-1 cut with Afe1-expected 3.8, 0.9, and 0.28 bands
• 6. A-2 cut with Afe1-expected 3.8, 0.9, and 0.28 bands
• 7. C-1 cut with Kpn1-expected 2.9, 0.25 bands
• 8. C-2 cut with Kpn1-expected 2.9, 0.25 bands
• 9. C-1 cut with Nco1-expected 1.7, 1.4 bands
• 10. C-2 cut with Nco1-expected 1.7, 1.4 bands

Results: All expected bands are present in all samples. A faint unexpected band is present at ~2.5-3.0 kb in lanes 3, 4, 5, 6, maybe 7 and 8. Band is not strong enough to be a full piece of the plasmid

Gel picture:

Note: Kept copies 6A1-2 (glycerol box A-3) and 1C3-1 (glycerol box A-4), discarded others. TJ Ciesla and Charlie Cooper streaked a plate from 1C3-1 for use later

Ligation-scale restriction digest of pSB1C3-BBa_J04450 and 6/6/14 PCR product

• EcoRI-HF and SpeI-HF (in Cutsmart buffer)
• 35 uL pSB1C3 DNA (337.2 ng/uL) in 50 uL reaction for 3 hrs
• 35 uL Repeat-gRNA-Repeat PCR product in 50 uL reaction for 3 hrs

Made 0.8% agarose TAE gel to separate pSB1C3 backbone from insert

Ran pSB1C3-BBa_J04450 digest on gel

• Expected to see two bands, 2.0 kb and 1.1 kb.

Results: only one large band ~3.0 kb with faint bands slightly below

Gel photo:

Analytical Restriction Digest

• To test why digest 6/9/14 did not work as expected
• pSB1C3-BBa_J04450 in three cuts, with pCC415 as a control cut (see gel results below)

Agarose gel of restriction digest

• 1. pCC415 cut with SacI-HF: expecting two bands
• 2. pSB1C3-BBa_J04450 cut with SacI-HF and SpeI-HF: expecting 2.0 and 1.1 kb bands
• 3. pSB1C3-BBa_J04450 cut with SacI-HF and EcoRI-HF: expecting 2.3 and 0.9 kb bands
• 4. pSB1C3-BBa_J04450 cut with SpeI-HF and EcoRI-HF: expecting 2.0 and 1.1 kb bands

Results: Expectations met

Gel photo:

Isolate DNA from restriction digest of PCR product (Repeat-seq-Repeat)

• DNA isolated using Qiagen Spin columns - last few steps of miniprep protocol
• DNA is 75 ng/µL in 30 µL EB buffer

Grow bacteria in medium

• 1. DH5α (Charlie’s) in SOC for future transformation
• 2. DH5α, plate 3, 15B in LB+CM* (pSB1C3-BBa_J06702)
• 3. DH5α, plate 2, 6F in LB+CM* (pSB1C3-BBa_R0040)
• 4. DH5α, plate 2, 17M in LB + CM* (pSB1C3-BBa_K741002)
• 5. DH5α, pSB1C3-BBa-J04450 in LB + CM**
• 6. DH5α, pSB6A1-BBa-J04450 in LB + amp**
• *2, 3, 4 contain plasmids w/biobricks
• **5 and 6 will be used to calibrate flow cytometry in 6/11/14

Next steps:

• Miniprep, digest to confirm plasmids from 2, 3, 4
• J06702 contains RBS-mCherry-T, to be PCRed out
• R0040 contains pTet, to be PCRed out
• K741002 contains pLac-RBS-GFP, and Terminator, both to be PCRed out
• Dilute 5 and 6, use for flow calibration

Froze 6 1mL seed stocks of DH5alphas in 15% glycerol

• From 6/10 culture
• For use in iGEM Chemically Competent Cell protocol

Grow 2 seed stocks in SOC overnight for CCEC protocol

• Each stock 1mL DH5alpha in 250mL SOC
• In shaker at room temperature (started 6:30pm)

Next Steps:

• Complete protocol with cultures

Froze 15% glycerol stocks of three biobrick plasmids at -80C

• DH5alpha, pSB1C3-BBa_K741002, stock location Box 1-A5
• DH5alpha, pSB1C3-BBa_J06702, stock location Box 1-A6
• DH5alpha, pSB1C3-BBa_R0040, stock location Box 1-A7

Miniprepped three plasmids

• Standard Qiagen kit protocol
• Results:
• 1C3-K741002 40uL at 98 ng/uL, iGEM box 1 A5
• 1C3-J06702 40uL at 114 ng/uL, iGEM box 1 A6
• 1C3-R0040 40uL at 170 ng/uL, iGEM box 1 A7

Analytical restriction digest of minipreps to confirm

• Each of three plasmids in EcoRI-HF/SpeI-HF and in PstII-HF
• All in cutsmart buffer, 2 hrs at 37C

Agarose gel of restriction digest

• 1. 1C3-K741002 in EcoRI-HF/SpeI-HF : expected 2.0, 1.1 kb bands
• 2. 1C3-K741002 in PvuII-HF : expected 1.9, 0.89, 0.4 kb bands
• 3. 1C3-J06702 in EcoRI-HF/SpeI-HF : expected 2.0, 0.89 kb bands
• 4. 1C3-J06702 in PvuII-HF : expected 2.0, 1.0 kb bands
• 5. 1C3-R0040 in EcoRI-HF/SpeI-HF : expected 2.0, 0.08 kb bands
• 6. 1C3-R0040 in PvuII-HF : expected 2.1 kb band

Results: Order of tubes in gel was not recorded. Lanes probably good, but should retest to be sure.

Gel photo:

Next steps:

• Reconfirm plasmids
• PCR pLac-RBS-GFP from K741002
• PCR RBS-mCherry-Term from J06702
• Isolate pSB1C3
• SLIC to make pDGC3

Diluted E. coli to grow to log phase for flow

• DH5alpha (SOC), pSB1C3-BBa_J04450 (LB+Cm), and pSB6A1-BBaJ04450 (LB+Amp)
• Each strain three dilutions: 1/100, 1/250, 1/500
• Growing from ~10am
• Note: Overnight culture of 1C3 had no visible red, but culture of 6A1 was strong reddish-pink. Possible that RFP is not expressed well in 1C3.

Flow Cytometry Parameter setting

• 1. calibration beads
• 2. messing around with parameters
• 3. pSB6A1-BBa_J04450 1/500 initial dilution
• 4. pSB1C3-BBa_J04450 1/500 initial dilution, 1/50 final dilution at flow time
• 5. DH5alpha 1/500 initial dilution, 1/50 final dilution at flow time
• 6. pSB6A1-BBa_J04450 1/100 initial dilution, 1/50 final dilution at flow time
• 7. pSB1C3-BBa_J04450 1/100 initial dilution, 1/50 final dilution at flow time

Notes: pSB1C3-BBa_J04450 showed strong, clear red fluorescence at both dilutions. pSB6A1 showed a bimodal fluorescence with some cells matching 1C3 fluorescence and others matching DH5alpha natural fluorescence. All samples showed two peaks of FS:SS data. These peaks did not correlate with the bimodal fluorescence seen in 6A1.

Parameters settled on (subject to change): FS: 350 volts, log 3 SS: 350 volts, log 3 Y2: 400 volts, log 5

Next Steps:

• Perform formal data analysis

G-block arrived today, CC split with Sargis

PCR of G-block to obtain iGEM piece

• Used oligos pSB1C3-up and pSB1C3-down
• iGEM protocol with 65C anneal, 20 sec extension

Agarose Gel of PCR product

• Used HindIII ladder instead of 2-log
• Results looked good, hard to tell exactly with ladder
• Some junk in no template control, but absent in other tubes

Gel photo:

PCR cleanup of G-block PCR

• Results: 30 uL product at 167.6 ng/uL

SLIC of pSB1C3 and Csy4-gRNA-Csy4 and Transformed

• 1 uL pSB1C3 from gel, purified (today), cut with EcoRI/SpeI
• 0.2 uL g-block in 10 uL reaction
• One no-backbone control (not enough backbone for no-insert control
• Transformed by heat shock into Charlie’s CCEC, plated on LB+Cm

Gel clean-up of pSB1C3 cut from EcoRI/SpeI gel

Gel picture:

• Digested and cut 6/10 and frozen overnight
• Used Zymoclean prep kit
• Results: Obtained 10 uL product at 133.1 ng/uL

Ligation of pSB1C3 with Repeat-seq-Repeat and Transform

• 4 uL pSB1C3 backbone from gel purified today
• 2 uL Repeat-seq-Repeat insert cut 6/9, cleaned 6/10
• One no-insert control (next time do a no-backbone control as well)
• Transformed by heat shock into Charlie’s CCEC, plated on LB+Cm

Results of transformation: no colonies on SLIC or Ligation plate

• Controls also blank
• Made new backbone for use (see below)

Overnight ligation of Repeat-seq-Repeat PCR with pSB1C3/p>

• ~100 ng backbone (2 uL) with ~30 ng insert (0.5 uL)
• in 20 uL reaction
• Two controls: one with no backbone, one with no insert
• Left in heat block in cold room (16C) at 6pm for overnight ligation

Next steps:

• Do a parallel room temp. ligation
• SLIC of Csy4 construct
• Transform all of the above, use Charlie’s CCEC until ours are confirmed

Restriction digest of pSB1C3-K741002 and pSB1C3-J06702

• 25 uL each backbone in 100 uL reactions
• SpeI-HF and EcoRI-HF in Cutsmart buffer (11am start)

Agarose gel of product for extraction

• One lane looked as expected (2.0 and 0.8 kb bands)
• Other lane had 2.0 kb backbone band but no insert band
• possibly a faint band ~.1 kb
• Extracted backbone (2.0 kb band) from both lanes separately

Gel photo:

Gel purification of pSB1C3

• Used Zymoclean prep kit
• Final elution:
• Confirmed piece 20 uL at ng/uL (teal tube)
• Unconfirmed piece 20 uL at ng/uL (orange tube)
• Notes: it is possible that the lack of insert is due to mislabelled tubes. Analytical digests and reculturing of the three plasmids in pSB1C3 (K741002, J06702, R0040) will determine if this is the case and correct any mislabelling.

PCR of pLac-RBS-GFP from pSB1C3-K741002

• With oligos pSB1C3-up and GFP-A-Z-3p
• Four tubes with 0, 0.4, 0.7, and 1.0 uL template (diluted to 1 ng/uL)
• iGEM protocol with 30 sec extension, 65C anneal

PCR of RBS-mCherry-Term from pSB1C3-J06702

• With oligos 5p-Z-Asc1-RBS-mCherry
• Four tubes with 0, 0.4, 0.7, and 1.0 uL template (diluted to 1 ng/uL)
• iGEM protocol with 30 sec extension, 67C anneal

Agarose gel of PCR results

• Lanes 1-4: pSB1C3-K741002 PCR
• No product (fuzzy primer bands only)
• Lanes 5-8: pSB1C3-J06702 PCR
• Some product present at low concentrations

Gel photo:

PCR cleanup of pSB1C3-J06702 PCR

• To see if low concentrations are enough to work with
• <50 ng/uL present, need to redo PCR
• Next steps: Redo PCR (once proper plasmids are confirmed), confirm, cleanup, SLIC

At ~11am, OD600 absorption rate for the cultures was .127 and .137

• Aiming for OD600 of 2-3
• Let grow for 50 more minutes (approx. one doubling time)
• OD600 still low (.163, .178), let grow another hour
• OD600 >0.2

Completed protocol as stated from iGEM website

• Used conical-bottom tubes instead of flat-bottom, resulting in difficulty of resuspending cell pellets. Had to vortex pellets substantially to resuspend, which may reduce competence.
• 50-ml tubes used were smaller than those used by iGEM headquarters. Result was more tubes, each with suggested volume, so final culture was too dilute (OD < .1). Pelleted and resuspended in half the volume
• Final volume 50 uL (for a double batch), in 50 tubes
• Tubes in -80C new box, “iGEM chemically competent cells”

Next steps: Test competence with transformation of known circular plasmid

Streaked single plate with frozen stocks of three plasmids in pSB1C3

• K741002, J06702, R0040
• All on same LB+Cm plate, because it was the last one
• Need to make more
• Incubated overnight at 37C

Next steps:

• Culture, miniprep and digest to confirm correct insert
• Digest current miniprep DNA as well
• Re-label tubes if necessary

Objective: Reconfirm plasmids in pSB1C3 backbone (possible mislabelling event)

Analytical Restriction Digest of 3 plasmids (2 digests each)

• Each plasmid in PvuII-HF and in EcoRI-HF/SpeI-HF
• All reactions in cutsmart buffer
• Note that plasmid names are as labelled, might not be what is present

Agarose gel of results:

1. pSB1C3-BBa_K741002 tube in PvuII-HF

2. pSB1C3-BBa_J06702 tube in PvuII-HF

3. pSB1C3-BBa_R0040 tube in PvuII-HF

4. pSB1C3-BBa_K741002 tube in EcoRI-HF/SpeI-HF

5. pSB1C3-BBa_J06702 tube in EcoRI-HF/SpeI-HF

6. pSB1C3-BBa_R0040 tube in EcoRI-HF/SpeI-HF

Results: No DNA in all K74 and J06 tubes. Tubes labelled R0040 appear to match expected for J06702

• K74 and J06 were 1 ng/uL dilutions, so DNA was likely too dilute to show up

• Hypothesis: R0040 and J06702 tubes are flipped. Possible that K741002 tube is incorrect as well
• gel picture:

Objective: Create pDGC3 using biobrick parts

PCR of pLac-RBS-GFP from BBa_K741002

• 8 tubes, with two templates in 4 tubes each (0, .4, .7, 1.0 uL template)

• “K” template is tube labelled as BBa_K741002 (1 ng/uL dilution)
• “J” tube is tube labelled as BBa_J06702 (1 ng/uL dilution)

• Trying in case this tube is mislabelled and contains K74

• Oligos pSB1C3up and GFP-A-Z-3p
• iGEM protocol with 30 sec extension time and 65C anneal

PCR of RBS-mCherry-Term from BBa_J06702

• 4 tubes at 0, .4, .7, and 1.0 uL template

• Used tube labelled “R” as template

• Suspect that this tube is mislabelled and contains J06

• Oligos 5p-Z-AscI-RBS-mCherry and pSB1C3-down
• iGEM protocol with 30 sec extension time and 67C anneal

Agarose gel of PCRs

Lanes 1-4: K741002 PCR with “K” template

Lanes 5-8: K741002 PCR with “J” template

Lanes 9-12: J06702 PCR with “R” template

• Results: primer bands only on all K741002 lanes. Strong expected band in lanes 10-12 indicating succesful PCR of J06702

• Further evidence that “R” is mislabelled and contains “J”

gel picture:

PCR cleanup of J06702 PCR

Objective: Remove BbsI site from mCherry in J06702

Restriction digest of tube labelled R0040

• Hopefully tube contains J06702 as predicted earlier
• With Bbs1 in buffer 2.1 with BSA

• 50 uL reaction with 25 uL DNA

Agarose gel of restriction digest

• Results: Looks as expected, hard to tell with single band
• gel photo:

PCR cleanup of Restriction digest

• Eluted in 30 uL
• Still need to check concentration
• Frozen at -20C

Objective: Create Repeat-seq-Repeat construct in pSB1C3

Ligation of Repeat-seq-Repeat PCR to pSB1C3 (confirmed green tube)

• Three identical tubes: “controls” actually have all pieces in them
• 20 uL reactions with 2 uL (100 ng) backbone and 0.5 uL (30 ng) insert
• Room temp for 1 hour

Transformation of ligations from overnight (6/12) and rt (6/13)

• 6 total tubes: 4 experimental and 2 controls (both from overnight)

Objective: Reconfirm and miniprep from frozen stocks of 3 plasmids in pSB1C3

Cultured 2 colonies each from 3 plasmid strains on 6/12/14 plate

• pSB1C3-K741002, pSB1C3-J06702, and pSB1C3-R0040

TODO for weekend:

• Miniprep cultures from frozen stock reconfirmations (Sat)
• Pick and culture colonies from 6 ligation plates (Sat)
• Miniprep cultures from ligation plates (Sun)

Gblock SLIC in to pSB1C3 conducted by CBC

• Objective: gRNA scaffold for 1) submission as a biobrick and 2) use for generating Csy4/ gRNA arrays

	backbone + insert	bb only	io
digested & purified backbone at ~50ng/uL	2	2	0
PCRd gBlock	1	0	1
buffer 2	1	1	1
10x BSA	1	1	1
water	5	6	7

• all but enzyme pre-mixed in 500uL eppies. enzyme added and incubate 2.5m at RT before icing tubes for 10m. 65 uL CBC’s chem. comp DH5alpha then 20m on ice, 45s 42C shock, 2m on ice, 60m recovery on SOC then spread on LB+Cm overnight.
• Results: no colonies the next day on any plates.

SLIC repair of BbsI cut site in mCherry by CBC

• Objective: BbsI/golden gate compatible mCherry for gRNA or crRNA array assembly schemes

• 1 uL each of mCherry-Bbs1-left and mCherry-Bbs1-right at 100uM added to 500uL eppies and evaporated in SpeedVac for backbone + insert and insert-only control. placed on ice to pre-cool

	backbone + insert	bb only	io
bbsi-dig backbone (~5 0ng/uL)	2	2	0
oligos (evapd)	2	0	2
buffer 2	1	1	1
10x BSA	1	1	1
water	6	6	8

• backbone + insert and bb only reactions treated with T4 poly as one 2x volume, then at 2.5m at RT 10 uL transferred to pre-chilled backbone + insert tube with waiting oligos. insert only reaction run as DNA-free dummy reaction for 2.5m in enzyme then transferred to pre-chilled tube with oligos.
• 65 uL chemically competent DH5alpha (from cbc) added at 10m on ice, incubated 20m, then heat shocked 45m at 42C then 2m on ice again before recovering for 60m in SOC and spread on plates.
• Results: > 100 colonies on backbone-only control. improper spreading of backbone + insert makes it hard to tell, but there may be slightly more colonies there than on the bb-only.

Analytical restriction digest and agarose gel of plasmids

1. pSB1C3-BBa_K741002-1 in EcoRI-HF/SpeI-HF (expected 2.0, 1.0 kb bands)
2. pSB1C3-BBa_K741002-2 in EcoRI-HF/SpeI-HF (expected 2.0, 1.0 kb bands)
3. pSB1C3-BBa_J06702-1 in EcoRI-HF/SpeI-HF (expected 2.0, 0.9 kb bands)
4. pSB1C3-BBa_J06702-2 in EcoRI-HF/SpeI-HF (expected 2.0, 0.9 kb bands)
5. pSB1C3-BBa_R0040-1 in EcoRI-HF/SpeI-HF (expected 2.0, 0.1 kb bands)
6. pSB1C3-BBa_R0040-2 in EcoRI-HF/SpeI-HF (expected 2.0, 0.1 kb bands)
7. pSB1C3-BBa_K741002-1 in PvuII-HF (expected 1.8, 0.8, 0.4 kb bands)
8. pSB1C3-BBa_K741002-2 in PvuII-HF (expected 1.8, 0.8, 0.4 kb bands)
9. pSB1C3-BBa_J06702-1 in PvuII-HF (expected 2.0, 1.0 kb bands)
10. pSB1C3-BBa_J06702-2 in PvuII-HF (expected 2.0, 1.0 kb bands)
11. pSB1C3-BBa_R0040-1 in PvuII-HF (expected 2.1 kb band)
12. pSB1C3-BBa_R0040-2 in PvuII-HF (expected 2.1 kb band)

• Results: All lanes look as expected. Confirmation that stocks are correct.
• Previous minipreps and other tubes resulting from unconfirmed versions of these plasmids have been thrown out. The one exception is the successful PCR of RBS-mCherry-Term from BBa_J06702
• Gel picture:

Objective: Obtain pSB1C3 backbone for various transformations

Prep-scale digest of pSB1C3-BBa_K741002

• Using XbaI and SpeI-HF in cutsmart buffer
• 45 uL template in 100 uL reaction

Prep-scale digest of pSB1C3-Bba_R0040

• Using EcoRI-HF and SpeI-HF in cutsmart buffer
• 45 uL template in 100 uL reaction

Gel extraction of digests to obtain pSB1C3 backbone

• Cut top band from K741002(left on picture)
• 240mg gel, eluted in 20uL H2O
• Cut only visible band from R0040(right on picture)
• Cleaned in 2 tubes(200mg each), eluted in 10+10=20uL H2O
• Cleaned up using Zymoclean prep kit
• Used 300uL wash buffer instead of 200uL for extra clean
• Gel picture:

Objective: Remove BbsI site from mCherry

SLIC transformations from 6/15(Charlie) showed colonies on the experimental plate as well as on the no-insert control

Cultured 6 colonies from plate for miniprep

• Each in 5mL LB+Cm at 37C
• In hopes of getting at least one mutated sequence

Cut BBa_J06702 with BbsI to try assembly again

• 4-hour digest with BbsI in NEBuffer 2.1 and BSA
• 45uL template in 100uL reaction
• Gel picture:

PCR cleanup of BbsI cut BBa_J06702

• Qiagen kit
• Final concentration 83.9 ng/uL

Objective: Create pDGC3 construct

PCR of plac-RBS-GFP-A-Z from BBa_K741002

• Used fresh Q5 polymerase and buffer
• template pSB1C3-BBa_K741002 diluted to 1 ng/uL
• 4 tubes with 0, 0.4, 0.7, and 1.0 template
• Oligos pSB1C3-up and GFP-A-Z-3p
• iGEM protocol, extension time 30 sec, 65C anneal temp

Agarose gel of PCR product

• Results: PCR appears to have worked (expected band size 1.0 kb)
• Gel picture:

PCR cleanup

• Qiagen kit
• Final concentration 161.1 ng/uL in 30 uL

TODO:

• SLIC of pSB1C3 SpeI/XbaI cut with G-block
• SLIC of pSB1C3 J06702 BbsI cut with oligos for mutation
• SLIC of pSB1C3-SpeI/XbaI cut with GFP PCR and mCherry PCR
• Ligation of pSB1C3 SpeI/EcoRI cut with Repeat-seq-Repeat PCR
• Possibly try Gibson of G-block assembly as well
• Possibly transform biobrick 4-4G (Bba_B0030 in pSB1C3)
• As a way to get pSB1C3 without gel extractions
• Miniprep cultures of mCherry possible mutants
• Cut with BbsI and another enzyme to test for successful mutants

Miniprep six colonies containing pSB1C3-BBa_J06702 with possible ∆BbsI

• Qiagen kit
• Concentrations (1-6): 284.5, 257.8, 268.2, 255.9, 242.2, 257.1 ng/uL

Analytical restriction digest and agarose gel of six possible ∆BbsI mutants

1. pSB1C3-J06702 (nonmutant control) cut with EcoRI-HF
2. pSB1C3-J06702 (nonmutant control) cut with BbsI/EcoRI-HF
3. pSB1C3-J06702 B1 cut with EcoRI-HF
4. pSB1C3-J06702 B1 cut with BbsI/EcoRI-HF
5. pSB1C3-J06702 B2 cut with EcoRI-HF
6. pSB1C3-J06702 B2 cut with BbsI/EcoRI-HF
7. pSB1C3-J06702 B3 cut with EcoRI-HF
8. pSB1C3-J06702 B3 cut with BbsI/EcoRI-HF
9. pSB1C3-J06702 B4 cut with EcoRI-HF
10. pSB1C3-J06702 B4 cut with BbsI/EcoRI-HF
11. pSB1C3-J06702 B5 cut with EcoRI-HF
12. pSB1C3-J06702 B5 cut with BbsI/EcoRI-HF
13. pSB1C3-J06702 B6 cut with EcoRI-HF
14. pSB1C3-J06702 B6 cut with BbsI/EcoRI-HF

• Expected single band at 3.0 kb in EcoRI cuts
• Successful mutants expected BbsI/EcoRI cuts to match EcoRI-only cuts
• Unsuccessful nonmutants expected 2.5, 0.5 kb bands in BbsI/EcoRI cuts
• Results: All six colonies unsuccessful (two bands in BbsI/EcoRI cut)
• All controls looked as expected
• gel picture:

Objective: Create various plasmids by Ligation/SLIC

Ligation to create pSB1C3-Repeat-seq scaffold

• 100 ng pSB1C3 cut with EcoRI/SpeI = 0.4 uL @ 30 ng/uL
• 30 ng Repeat-seq PCR = 0.4 uL @ 77 ng/uL
• No-backbone and no-insert controls

SLIC to create pSB1C3-Csy4-gRNA-scaffold

• 100 ng pSB1C3 cut with SpeI/XbaI = 1.1 uL @ 91 ng/uL
• 30 ng (3xmolar) G-block PCR = 0.2 uL @ 167 ng/uL
• No-backbone and no-insert controls

SLIC to create pDGC3 (with GFP and mCherry in pSB1C3)

• 100 ng pSB1C3 cut with SpeI/XbaI = 1.1 uL @ 91 ng/uL
• 150 ng (3xmolar) GFP-A-Z PCR = 2.5 uL @ 61 ng/uL
• 150 ng (3xmolar) mCherry PCR = 1.1 uL @ 132.8 ng/uL
• No-insert control (no-backbone shared with Csy4 SLIC)

SLIC to remove BbsI from pSB1C3-J06702

• 100 ng pSB1C3-J06702 cut with BbsI = 1.2 uL @ 83.9 ng/uL
• Oligos mCherry-BbsI-left and mCherry-BbsI-right (0.5 uL each)
• No insert and no-backbone controls

Notes:

• SLICs may have been left on ice too long (>10 mins) before cells were added
• Large number of tubes for pipetting made timing uneven
• All 11 samples plated on LB+Cm plates

Objective: Test CCEC from 6/12/14

Transformed uncut plasmid (pSB1C3-J06702-B5) with new CCEC

• 4 different concentrations (1/10 dilutions each):
• 200 ng, 20 ng, 2 ng, and 0.2 ng
• Each in 10 uL tubes
• Plated on LB+Cm

Charlie’s plate protocol

• Mixed 1L Agar+LB, Autoclaved on liquid cycle
• Let cool while stirring, added 1x Chloroamphenicol
• Poured plates, let solidify

Objective: Check and culture from transformations 6/17

Results:

• Ligation of Repeat-seq construct to pSB1C3
• No colonies on experimental or either control
• SLIC of G-block (Csy4 construct) into pSB1C3
• No colonies on experimental or either control
• SLIC of pDGC3 (pSB1C3-pLac-GFP-mCherry)
• ~10 colonies on experimental plate
• ~6 colonies on Insert-only control, none on Backbone-only
• SLIC of pSB1C3-J06702 ∆BbsI
• ~100-200 colonies on experimental plate
• ~60 colonies on Backbone-only control, none on Insert-only
• CCEC Test
• Mat of colonies covering plates with 200ng, 20ng, and 2ng plasmid
• ~2000 colonies covering plate with 0.2ng plasmid
• Results: competency 10,000 colonies/ng (high success rate)

Culture colonies overnight from successful SLICs

• 6 cultures from pDGC3 (pSB1C3-pLac-GFP-mCherry) (labelled 1-6)
• 6 cultures from pSB1C3-J06702-∆BbsI (labelled 7-12)
• All in LB+Cm at 37C

Objective: Build pSB1C3-Repeat-crRNA-Repeat and pSB1C3-rCsy4-sgRNA-rCsy4)

PCR of Repeat-crRNA-Repeat from pdCas9

• Oligos 5p-EcoRI-XbaI-Repeat and SpeI-Repeat-3p
• Using iGEM stock of Q5 polymerase and buffer
• 4x50uL reactions, with template 0, 0.4, 0.7, and 1.0 uL
• 30sec extension, 63C anneal

PCR of G-block (rCsy4-sgRNA-rCsy4)

Oligos pSB1C3-up-1 and pSB1C3-down-1

Using iGEM stock of Q5 polymerase and buffer

4x50uL reactions, with template 0, 0.4, 0.7, and 1.0 uL

Left at -20C for ~1.5 hrs before run because thermocycler in use

30sec extension, 66C anneal

Agarose gel of both PCR products

• Lanes 1-4: Repeat-crRNA-Repeat PCR
• Lanes 5-8: rCsy4-sgRNA-rCsy4 PCR
• Results: strong bands in all six experimental tubes.
• Smear upward could indicate problems, but probably correct
• Control for Csy4 (Lane 5) alone showed strong band at 1kb, no explanation

gel picture:

PCR cleanup of both PCR products

• Qiagen kit
• Final concentrations in Elution Buffer (via nanodrop):
• Repeat-crRNA-Repeat PCR 30 uL at 131.7 ng/uL
• rCsy4-sgRNA-rCsy4 PCR 30 uL at 202 ng/uL

Prep-scale restriction digest of pSB1C3 backbone

• Used 70 uL combined from tubes of pSB1C3-J06702-∆BbsI (6/17)
  • Shown to be without mutation on 6/17
• XbaI/SpeI-HF in cutsmart buffer for 4 hours
• extracted larger (2.0kb) band from agarose gel

gel picture:

Gel cleanup of pSB1C3 backbone

• Used Qiagen kit instead of Zymoclean (first time)
• No isopropanol or sodium acetate necessary
• 340 mg of gel eluted into 30 uL Elution Buffer
• Final concentration 235 ng/uL (via nanodrop)

Gibson Assembly of two plasmids

• pSB1C3-rCsy4-sgRNA-rCsy4
• 100 ng = 0.42 uL pSB1C3 backbone cut with Xbal/SpeI
• 50 ng = 5xMolar = 0.25 uL G-block PCR
• pSB1C3-Repeat-crRNA-Repeat
• 100 ng = 0.42 uL pSB1C3 backbone cut with Xbal/SpeI
• 50 ng = 5xMolar = 0.38 uL Repeat-seq PCR
• Backbone-only control, insert-only controls for each reaction, positive control
• Transformed using iGEM CCEC, Plated on LB+Cm

Miniprep Colonies

• Qiagen Set
• Concentrations of ∆Bbs1 site mutation (7-12) (ng/μL): 192.6, 235.7, 279.3, 239.8, 238.6, 203.8
• Concentrations of pDGC3 (1-6) (ng/μL): 229.6, 301.9, 202.3, 206.6, 225.5, 259.1

Test Plasmids with Gel Digest

• Gel 1: log-2 ladder
• Gel 2-7: ∆Bbs1 site mutation 7-12 tested with EcoR1-HF
• Gel 8-13: pDGC3 1-6 tested with Spe1-HF
• Gel 14-19: ∆Bbs1 site mutation 7-12 tested with EcoR1-HF and Bbs1
• Gel 20-25: pDGC3 1-6 tested with Asc1 and Nhe1-HF

gel picture:

Objective: Observe Ligation Colony Results and Culture Test Colonies

Observe Plates

• Csy4-sgRNA-Csy4 Scaffold: 6 colonies
• Repeat-seq-Repeat Scaffold: 7 colonies
• Gibson positive control: 6 colonies
• Backbone-only: 1 colony
• Insert only: No colonies

Culture Test Colonies

• Cultured 3 Cys4-sgRNA-Cys4 Scaffold colonies
• Cultured 3 Repeat-seq-Repeat Scaffold colonies

Miniprep Colonies

• Qiagen Set
• Concentrations of Cys4-sgRNA-Cys4 Scaffold (1-3) (ng/μL): 168.4, 165.9, 220
• Concentrations of Repeat-seq-Repeat Scaffold (1-3) (ng/μL): 216.9, 84.0, 226.9

Test Plasmids with Gel Digest

• Gel 1: log-2 ladder
• Gel 2-4: Repeat-seq-Repeat Scaffold 1-3 tested with Acs1 and Nhe1-HF
• Gel 5-7: Cys4-sgRNA-Cys4 Scaffold 1-3 tested with EcoR1-HF and Bbs1
• Gel 8-10: pDGC3 1-6 tested with EcoR1-HF and Bbs1
• Gel 11-13: pDGC3 1-6 tested with EcoR1-HF and Mfe1-HF

gel picture:

Objective: Remove Bbs1 site from mCherry using SLIC

Prepare and Dephosphorylate Bbs1-cut mCherry-pSB1C3

• Used official NEB protocol of Calf intestinal protein on Bbs1-cut mCherry-pSB1C3 plasmid
• Purified DNA with miniprep with a concentration of 27 ng/μL

SLIC out the Bbs1 site using change oligos

• SLIC performed as per standard protocol with the mCherry-∆Bbs1-up and mCherry-∆Bbs1-down oligos on prepared plasmid
• Backbone only and insert only controls prepared

Transform into Chemically competent cells

• Heat shocked and incubated, then plated as per standard protocol

Objective: Create pDGC3 with Gibson Assembly

Perform Gibson Assembly to create pDGC3 plasmid

• Gibson assembly done with pSB1C3 backbone and GFP and mCherry PCR-amplified inserts
• Backbone-only, insert-only and positive controls also prepared
• Gibson master mix not included on insert-only

Transform assemblies into cells

• Heat shocked and incubated, then plated as per standard protocol

Took plates out of incubator and left at room temperature

Observe plates

• DESCRIPTION?
• Choose four colonies to grow over

Incubate Colonies

• Colonies grown in LB+Cm overnight

Objective: Sequence rCsy4-sgRNA-rCsy4 and Repeat-seq-Repeat for BioBrick submission

Prepare plasmid submission using Big Dye protocol

• pSB1C3-up and pSB1C3-dn oligoes used
• Buchler thermal phaser used to run reaction

Deliver prepped plasmid to DUGSIM

• Delivered to lab room in BioSci

Next step: Compare sequence to predicted, submission process

Objective: Test if ∆Bbs1 site was removed

Miniprep Colonies

• Qiagen Set
• Concentrations of ∆Bbs1 mCherry-pSB1C3 (1-4) (ng/μL): 168.0, 110.2, 175.7, 147.5

Digest mCherry-pSB1C3 plasmid

• Standard diagnostic digest protocol used on four overnight colonies from 6/22
• Each colony had two digests: EcoR1 and EcoR1/Bbs1

Run and observe gels

• Gels run for 17 minutes
• Order: 2-log ladder, EcoR1-cut sample 1, EcoR1 and Bbs1-cut sample 1, EcoR1-cut sample 2, EcoR1 and Bbs1-cut sample 2, EcoR1-cut sample 3, EcoR1 and Bbs1-cut sample 3, EcoR1-cut sample 4, EcoR1 and Bbs1-cut sample 4

Objective: Insert sgRNA sequence into rCsy4-sgRNA-rCsy4 Scaffold

Cut Scaffold with Bsa1 enzyme

• 36 μL of DNA template was cut with 5 μL of enzyme in 10 μL of CutSmart buffer and 49 μL of water
• Digested for 2 hours

• Qiagen Set clean-up protocol performed
• Concentrations of rCsy4-sgRNA-rCsy4 Scaffold (ng/μL): 93.2

Anneal mCherry1 sgRNA from oligos

• crRNA-ch1-up and crRNA-ch1-dn oligos annealed using ____ protocol

Ligate into scaffold

• Ligate annealed oligos into the cut rCsy4-sgRNA-rCsy4 scaffold using ligation protocol

Insert Tet1 sgRNA into scaffold using SLIC

• Used standard SLIC protocol, using scaf-crRNA-Tet1-up and scaf-crRNA-Tet1-dn

Transform into competent cells

• Used iGEM stock comp cells
• Plated onto LB+Cm plates

Objective: Get pSB1C3 backbone from BBa_R0030

Awaken and Transform R0030 into cells

• BBa-R0030 awakened from frozen iGEM supply
• Transformed onto iGEM stock competent cells and plated onto LB+Cm

Objective: Check plates from 6/22/13

Ligation and SLIC of sgRNA

• Backbone only and experimental plates have many colonies
• Theories are that Bsa1 has degraded or that the plasmid self-ligated
• Next step: test Bsa1

Objective: Test Bsa1 effectiveness

Digest test plasmids with Bsa1

• TAL 16C, 20A and 20B plasmids used (from last year’s stocks in iGEM Box 3)
• Standard analytical restriction digest used with Bsa1 in CutSmart
• Left for 2 hours in 37°C

Ran diagnostic gel

1. TAL 16C cut with BsaI (expected 2 fragments around 2.3 kb and one around 5.6 kb)

2. TAL 20A cut with BsaI (expected 2 fragments around 2.3 kb and one around 6.0 kb)

3. TAL 20B cut with BsaI (expected 2 fragments around 2.3 kb and one around 6.0 kb)

• Results: Bsa1 does cut, but fragments were not exactly expected sizes

• 16C showed one smeary band ~ 2-3 kb plus a faint, thin band ~ 5-6kb
• 20A showed two clear bands at ~4-5 kb and ~2-3 kb

• Known to have issues, may be incorrect insert

• 20B showed two clear bands at ~6 kb and 2-3 kb

gel picture:

Objective: Attempt mCherry Bbs1 Mutation

Charlie Cooper tried today.

• BsmBI digest:

• 30uL of 159.1ng/uL pSB1C3-BBa_J06902 (mCherry) digested with BbsI in 100uL reaction & NEBuffer 2.1 for 4h237C. Collected with PCR cleanup protocol and eluted in to 80uL h2o. Ran 2uL on a gel next to undigested DNA at same final concentration. Untreated plasmid runs faster than BbsI-treated, consistent with behavior of un-digested circular DNA.
• 
• 1: ladder
• 2: digested plasmid
• 3: undigested
• 4,5 unrelated stuff for CBC

• CIP:

• Added 10uL buffer 3 and 2uL undiluted CIP. 37C for 60m. PCR cleanup again and eluted in to final volume of 35uL with conc ~76 ng/uL

• SLIC

• 1 tube with 1 uL each of SLIC oligos mCherry-Bbs1-left and -rightprepared by speedVac to remove solvent. tubes placed on ice.
• T4 reaction set up with 2 uL backbone (0 for insert-only), buffer, bsa and water to 10uL. 0.5 uL T4 poly added and incubated at RT. at 2.5m, tube contents transferred to waiting tube with (or without for bbb-only) dried oligos.
• 10m on ice, add 60uL comp. DH5alpha, 20m more on ice then 45s heat shock and 2m more on ice before recovering for 60m in SOC and spreading on LB+Cm and 37C overnight

Objective: Build pDGC3 construct

PCR of pLac-GFP-A-Z from pSB1C3-BBa_K741002

• 4 tubes with 0, 0.4, 0.7, 1.0 uL of 1 ng/uL template

• 6/16 dilution of 6/14 miniprep

• Oligos pSB1C3up and GFP-A-Z-3p
• iGEM stock of Q5 polymerase/buffer
• iGEM thermocycler protocol with 30 sec extension, 65C anneal

PCR of Asc1-mCherry-Term from pSB1C3-BBa_J06702

• 4 tubes with 0, 0.4, 0.7, 1.0 uL of 1 ng/uL template

• 6/16 dilution of 6/14 miniprep

• Oligos 5p-Z-AscI-RBS-mCherry and pSB1C3dn
• iGEM stock of Q5 polymerase/buffer
• iGEM thermocycler protocol with 30 sec extension, 67C anneal

Agarose gel of PCR products:

• Successful product in lanes 2 and 4 of GFP, and 2, 3, 4 of mCherry PCR

gel picture:

PCR cleanup of GFP and mCherry amplicons

• Accidentally pooled all four tubes of GFP together

• Should not be a problem, may reduce yield slightly

• Correctly pooled 3 tubes of mCherry
• Final concentrations:

Objective: Culture 16 colonies from various plates:

pSB1C3-BBa_B0030

• Transformed 6/23

• Plate had >100 colonies

• Cultured 1 in 5mL LB+Cm

• Next steps: use to extract pSB1C3 backbone without gel purification

Gibson products of pDGC3 assembly

• Transformed on 6/20
• Backbone+Insert plate had ~75 colonies

• Cultured 3 in 5mL LB+Cm

• Insert-only control plate had ~200-300 colonies

• Cultured 2 in 5mL LB+Cm

• Backbone-only control had ~15-20 colonies

• Cultured 2 in 5mL LB+Cm

• Next steps: analytical digests to test if any products were successful, and why insert-only products are so numerous

• Insight into how to move forward with troubling construct?

Ligation products of crRNA mCh1 into Repeat-seq scaffold

• Transformed on 6/23
• Backbone+Insert plate had hundreds of tiny colonies

• Cultured 3 in 5mL LB+Cm

• Backbone-only control had hundreds of tiny colonies

• Cultured 1 in 5mL LB+Cm

• Insert-only control had no colonies
• Next steps: Test to see if any products were successful, or which false construct caused backbone-only products

SLIC products of crRNA Tet1 into Repeat-seq scaffold

• Transformed on 6/23
• Backbone+Insert plate had hundreds of tiny colonies

• Cultured 3 in 5mL LB+Cm

• Backbone-only control had hundreds of tiny colonies

• Cultured 1 in 5mL LB+Cm

• Insert-only control had no colonies
• Next steps: Test to see if any products were successful, or which false construct caused backbone-only products

Objective: Find constitutive GFP constructs for Molecular Titration approach

Transformed four biobricks in pSB1C3 from iGEM kit plates

• Plate 1-5I:BBa_K608008

• “strong promoter/medium RBS + GFP”

• Plate 1-5K:BBa_K608010

• “medium promoter/strong RBS + GFP”

• Plate 1-5M:BBa_K608011

• “medium promoter/medium RBS + GFP”

• Plate 1-5O:BBa_K608012

• “medium promoter/weak RBS + GFP”

• Transformed by heat shock using iGEM CCEC stocks

• Plated on LB+Cm

Objective: Check plates from 6/24/13

Check ∆Bbs1 Plates

• Backbone only and experimental plates have many colonies, leading to possibility that CIP did not function

Objective: Check CIP still functions

Digest plasmid with EcoRI

• pSB1C3-J06702 ∆Bbs1 plasmids 8 and 9 were used
• Standard ligation prep digest preformed on 32 uL of template in CutSmart buffer
• Digest cleaned with Qiagen PCR clean-up into 30uL of EB
• Concentration of ∆Bbs1 8 was 99.2 ng/uL and 9 was 145.6 ng/uL.

Treat experimental group with Calf Intestinal Phosphotase

• 15 uL of the cleaned DNA included in standard CIP protocol
• CIP cleaned with Qiagen PCR clean-up into 15 uL of EB
• Concentration of ∆Bbs1 8 was 32.6 ng/uL and 9 was 62.2 ng/uL.

Ligate plasmids with T4

• Ligation protocol used triple amounts of all compounds as standard 100 ng ligations, producing 300 ng of ligated DNA for ∆Bbs1-8, ∆Bbs1-8-CIP, ∆Bbs1-9 and ∆Bbs1-9-CIP in 60 uL
• 40 uL was cleaned with a Qiagen PCR clean-up into 8.5 uL of EB
• 20 uL was left to transform into cells

Digest with EcoRV and analyze digestion

• 8.5 uL of DNA, since concentrations are so low, were used in a restriction digest with 1 uL of CutSmart and 0.5 uL of EcoRV
• Ran on gel to test digest
• Order: 2-log, ∆Bbs1-8, ∆Bbs1-8-CIP, ∆Bbs1-9, ∆Bbs1-9-CIP

Transform DNA into cells to see rate of uptake

• Transformed into iGEM comp cells and plated onto LB+Cm

Objective: Build crRNAs in Repeat-seq scaffold

Miniprep crRNA cultures and BO controls

• 3 copies crRNA-Tet1 and one BO control

• Concentrations in 50 uL (1,2,3,BO):

• 68, 124.7, 71.4, 89.2 ng/uL

• 3 copies crRNA-mCh1 and one BO control

• Concentrations in 50 uL (1,2,3,BO):

• 55.8, 51.3, 60.6, 65.1 ng/uL

• Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)

Analytical digest of crRNA-Tet1 minipreps

• To determine if minipreps still digestible (if in glycerol, may not be)
• 4 tubes digested with EcoRI-HF/EcoRV-HF in cutsmart buffer for 2 hrs

Agarose gel of digest results:

1. crRNA-Tet1-1 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

2. crRNA-Tet1-2 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

3. crRNA-Tet1-3 in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

4. crRNA-Tet1-BO in EcoRI-HF/EcoRV-HF (expected 1.3 and 0.9 kb bands)

• Results: expected bands present, minipreps still useful in digests

Analytical digest of crRNA minipreps

• To test whether new insert is present
• 8 tubes digested with BsaI/NcoI in cutsmart buffer for 2 hrs
• Frozen in iGEM freezer--need to run on gel

Objective: Build pDGC3

MIniprep pDGC3 cultures, BO controls, and IO controls

• 3 copies pDGC3, 2 copies BO control, 2 copies IO control

• Concentrations in 50 uL (1,2,3,BO-1,BO-2,IO-1,IO-2):

• 55.6, 66.9, 54.1, 32.1, 36.2, 55.5, 58.7 ng/uL

• Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)

Analytical digest of minipreps

• To test whether GFP and/or mCherry are present
• Two digest each of 7 samples, NdeI/EcoRV-HF and BstEII-HF/EcoRV-HF
• All in cutsmart buffer for 2 hrs
• Frozen in iGEM freezer--need to run on gel

Objective: Obtain pSB1C3 backbone without gel purification

Miniprep pSB1C3-B0030 culture

• Concentration 34 ng/uL (eluted in 50 uL)
• Notes: unsure why concentrations are so low. Could have eluted in 50% glycerol instead of water (bottles look similar)

Prep-scale digest of pSB1C3-B0030 to obtain backbone

• Enzymes SpeI-HF/EcoRI-HF in cutsmart buffer
• 42 uL DNA in 100 uL reaction for 4 hrs
• Note: product frozen in iGEM cloning project box overnight
• Need to run on gel to confirm and PCR clean to use

Objective: Culture colonies containing constitutive GFP for molecular titration approach

• pSB1C3-B0030(as a control with no flourescence)
• pSB1C3-K608008“strong promoter/medium RBS + GFP”
• pSB1C3-K608010“medium promoter/strong RBS + GFP”
• pSB1C3-K608011“medium promoter/medium RBS + GFP”
• pSB1C3-K608012“medium promoter/weak RBS + GFP”
• To flow tomorrow and save glycerol stocks

Objective: Check plates from 6/25/13 to confirm CIP activity

Check ∆Bbs1-8 and 9 Plates

• Still many colonies on CIP treated plates, but much fewer than non-CIP treated plates
• This leads to the conclusion that CIP still functions, but is not completely effective, leading to possible expression of self-ligation in CIP treated ∆Bbs1 experiments

Objective: Dephosphorylate Bbs1 cut pSB1C3-J06702 (mCherry)

Run dephosphorylation

• 3uL of Cutsmart and 1.5 uL of CIP added to 25 uL of mCherry plasmid, set in 37C for 45 minutes
• DNA cleaned with Qiagen PCR Clean-up

Objective: Analyze the constitutive GFP expression of reporters using flow cytometry

Save stocks of Constitutive GFP plasmids

• K608008, K608010, K608011, K608012

Flow Cytometry

• FSC log 3, 350V
• SSC log 3, 350V
• B1 log 5, 350V
• Order of samples:

1. B0030 (control)

2. K608008

3. K608010

4. K608011

5. K608012

Results: (analyzed 6/30/14)

	E. coli/Simple peak GFP | Mean (B1-A)	Fold Change from control
pSB1C3-B0030 (control)	0.29	1
pSB1C3-K608008	0.3	1.03
pSB1C3-K608010	201	693.10
pSB1C3-K608011	58.2	200.69
pSB1C3-K608012	118	406.90

Notes:

• K608008 showed no fluorescence--could be transformation problem or part problem.
• K608010 had messier data for FSC/SSC and for GFP (B-1). Despite having the highest mean, this is not as reliable of a sample.
• K608012 had the highest reliable mean, and should be used for at least the initial molecular titration experiments. K608011 is also useful if we need a lower maximum fluorescence level.

Objective: Insert crRNAs targeting GFP into pdCas9 and Repeat scaffold

Prep-scale Digest of pdCas9

• BsaI in Cutsmart buffer
• 30 uL template in 100 uL reaction
• 4 hrs at 37C

Anneal crRNA inserts

• 3 inserts, each annealed with 2 oligos

• crRNAs GFP1, GFP2, and GFP3

• 5uL each oligo in ~boiling water bath, cooled at rt to rt

Ligation of crRNAs into BsaI site in pdCas9 and Repeat scaffold

• 6 reactions plus 5 controls
• pdCas9 with crRNA inserts GFP1, GFP2, GFP3 (annealed as above)

• backbone cut with BsaI (6/30), 100ng=3.5uL

• pSB1C3-Repeat-scaffold (pDGC1) with crRNA inserts GFP1, GFP2, GFP3 (annealed as above)

• backbone cut with BsaI (6/23), 100ng=1uL

• BO controls for pdCas9 and Repeat-scaffold
• IO controls for all three crRNA inserts
• 10 uL rxns transformed into DH5alphas (iGEM’s CCEC), plated on LB+Cm

Objective: Build pDGC3

Gibson assembly of pSB1C3, GFP, and mCherry

• Backbone cut with EcoRI/SpeI on 6/16, 100ng=1.5uL
• GFP PCR treated with DpnI on 6/26
• mCherry PCR treated with DpnI on 6/26
• Two copies, one with 100ng backbone, one with 50ng backbone

• 1xMolar and 2xMolar inserts (50ng each)

• BO and IO controls
• Using Mert’s new DIY Gibson mix (15uL into 20uL)
• Transformed into DH5alphas (iGEM’s CCEC), plated on LB+Cm

Objective: Remove BbsI site from mCherry

SLIC to remove BbsI site from pSB1C3-J06702

• Backbone pSB1C3-J06702 super-treated with CIP on 6/26, 100ng=2.5uL
• Oligos mCherry-BbsI-left and mCherry-BbsI-right
• 10 uL rxns transformed into DH5alphas (iGEM’s CCEC), plated on LB+Cm