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.

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