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

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)

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