Team:Columbia-Cooper-NYC/Columbia notebook 2

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Revision as of 20:17, 15 September 2012


Columbia Genetics Lab Notebook

July, 2012

Thursday, 5th

  • Re-hydrated plasmids with 50µl of LB and Kanamycin solution
  • Stored solution at 37°C incubator overnight

Friday, 6th

  • Purified pET26b vector using standard DNA purification protocol

Monday, 9th

  • Received kill gene Bba-K124017 from plate 3, 20M
  • Re-hydrated DNA according to standard iGEM re-hydration protocol

Tuesday, 10th

  • Contacted professors at Germany in hopes to receive copies of fungal phytochrome FphA

Wednesday, 11th

  • Received confirmation by professors at Germany for FphA to be sent to Columbia University
  • Conducted transformation using electroporation with competent bacteria (marked by resistance to Kanamycin)
    1. Control: 1µl of deionized water with abt. and 60µl of bacteria cells
    2. Variable: 1µl of re-hydrated kill gene and 60µl of bacteria cells
  • Placed both samples after electroporation into 200µl of preprepared LB
  • Placed samples in shaker 37°C for 30 minutes

Thursday, 12th

  • Grew 1 colony of transformed bacteria in 5mL of Kanamycin and LB solution
    • Note: Using pET20b vector over pET26b vector from glycerol stock solution

Friday, 13th

  • Isolated 4 samples of plasmid using standard plasmid isolation protocol
    1. 2 samples: kill gene
    2. 2 samples: pET20b vector

Monday, 16th

  • Re-hydrated two biobrick parts in plasmid pSB2K3 according to standard iGEM re-hydration protocol
    1. BBa-I16009 (PcyA) from plate 1, 20F
    2. BBa-I16008 (ho1) from plate 2, 13J
  • Electroporated 1µl of each biobrick into separate E. coli at 1800V
  • Added 100µl LB broth into each sample
  • Placed samples at 33.4°C for 20 minutes
  • Samples were plated to be grown overnight

Tuesday, 17th

  • Placed 5ml each of LB/Kan into two centrifuge tube for PCB creation
    1. Label P: PcyA
    2. Label h: ho1
  • Placed samples in 37°C incubator

Wednesday, 18th

  • Purified ho1 and PcyA plasmids using standard DNA purification protocol
  • Placed purified DNA into glycerol stock (LB/Kan) and stored at -80°C

Thursday, 19th

  • Purified GFP using standard DNA purification protocol
  • Prepared glycerol stock solution (500µl GFP/500µl 80% glycerol) and stored at -80°C

Tuesday, 24th

  • Re-hydrated four biobrick parts according to standard iGEM re-hydration protocol
    1. Inducible plasmid (pSB1AK3-J04500) from plate 4, 12A
    2. GFP (pSB1A2-E0040) from plate 1, 14K
    3. High copy plasmid pSB1T3-J044500 from plate 1, 7A
    4. Low copy plasmid (pSB3C5-J044500) from plate 1, 3C
  • Electroporated competent E. coli with each of the four above genes separetely
  • Created Kan, Amp, Cam, Tetra, and Amp/Kan plates

Wednesday, 25th

  • Streaked pSB1T3-J04450
  • Created LB solution with Kan or Amp or Cam

Thursday, 26th

  • Prepared Glycerol stock for inducible promoter, GFP, and low-copy plasmid
  • Picked a single colony from pSB1T3-J04450 and let it grow overnight in 37C
  • Recorded and measured the DNA concentrations of following at 260nm:
    1. Kill gene
    2. PcyA
    3. ho1
    4. GFP
    5. Inducible promoter
    6. Low copy CAM plasmid
  • Followed digestion and ligation protocol; setup explained below:
    1. Upstream: ho1; Downstream: PcyA; Destination plasmid: Low-copy CAM
    2. Upstream: Inducible promoter; Downstream: GFP; Destination plasmid: Low-copy CAM
    3. Upstream: Inducible promoter; Downstream: Kill; Destination plasmid: High-copy TET (pSB1T3)
  • After completion, store samples at -20C

Friday, 27th

  • Electroporated ligated samples from previous day: GFP, PCB, Kill
  • Followed standard plasmid isolation protocol for pSB1T3-J04450 (TET plasmid)

Saturday, 28th

  • Check plates from electroporation from previous day

Monday, 30th

  • Stored pif3 and phyB that arrived from Sweden
  • Relocated and reorganized the iGEM biobrick kit and glycerol stocks
  • Picked colonies for following DNA:
    1. Inducible promotor (pSB1AK3_J04500)
    2. GFP (pSB1A2_E0040)
    3. Low copy CAM plasmid (pSB3C5_J04150)
  • Added 10µl of ho1 and PcyA to 5ml of antibiotics
  • Electroporated following DNA:
    1. Inducible promoter IPTG/kill gene in BL21 cells
    2. Inducible promoter IPTG/GFP in BL21 cells
    3. PCB in α cells
  • Transferred successful electroporated cells to eppendorf tube with 100μL of LB and placed in 37C shaker

Tuesday, 31st

  • Prepared glycerol stock of the following:
    1. GFP
    2. Inducible promoter (IPTG)
    3. Low copy CAM plasmid
    4. pcyA
    5. ho1
  • Isolated ho1 and PcyA using standard plasmid isolation protocol

August, 2012

Wednesday, 1st

  • Applied IPTG to samples of bacteria with GFP or kill gene and placed back in incubator at 37C
  • Electroporated the following plasmids:
    1. IPTG inducible promoter/kill gene into BL21 cells
    2. IPTG inducible promoter/GFP into BL21 cells
    3. phyB into α select cells
    4. pif3 into α select cells
  • Followed the PCR purification protocol for samples 1, 2 listed above
  • Chemically transformed the following using standard heat shock protocol provided by Bioline:
    1. IPTG inducible promoter/kill gene into BL21 cells
    2. IPTG inducible promoter/GFP into BL21 cells
  • Plated PCB onto LB/CAM plate
  • Plated Pif3 and phyB on KAN plates
  • Plated 500μL of GFP containing cells among two plates each LB/CAM (with and without IPTG)
  • Placed all samples in 37C incubator

Thursday, 2nd

  • Selected colonies from PhyB from the LB/Kan plate
  • Prepared CAM plates
  • Selected colonies from GFP control
  • Received agar stabs from Uppsala

Friday, 3rd

  • Placed 60μL of IPTG to half of control plate for reconfirmation of results
  • Streaked GFP (with IPTG inducible promoter) and Pif3 on plates

Saturday, 4th

  • Streaked all 6 Uppsala (labelled below) parts from stabs onto plates:
    1. Upps 1: pSB1K3-B0034-YF1-B0034-FixJ
    2. Upps 2: pSB1K3-YF1
    3. Upps 3: pSB1K3-FixJ
    4. Upps 4: pSB1C3-PfixK2
    5. Upps 5: pSB1A3-amilCP
    6. Upps 6: pSB1C3 - amilGFP
  • Prepared glycerol stock for phyB (ETHZ)
  • Isolated phyB by following standard plasmid isolation protocol
  • Electroporated fphA and ho1 from Germany
  • Created LB/TET, LB/CAM, LB/Amp, LB/Kan plates

Sunday, 5th

  • Pulled colonies from 6 iGEM parts and ho1 from Germany
  • Streaked fphA from Germany
  • Pulled colonies for Upps 2, 3, 4, 5, 6
  • Streaked Upps 1, 3, 4, 6
  • Parafilmed all plates and placed in 4C
  • Sorted and threw out unnecessary plates

Monday, 6th

  • Pulled colonies from fphA and Upps 1
  • Isolated plasmids for the following samples following standard plasmid isolation protocol:
    1. 6 iGEM parts
    2. Upps 2-6
    3. ho1
  • Reconfirmed that TET plates are valid

Saturday, 11th

  • Reviewed the solutions for diluted GFP and observed no significant results
  • Reorganized samples in fridges and incubators

Monday, 13th

  • Chemically transformed competent cells (BL21) with plasmids below using bioline protocol (used 1/2 of recommended amount)
    1. IPTG-Upps 5-Low Copy (CAM)
    2. IPTG-Upps 6-Low Copy (CAM)
    3. IPTG-Kill gene-Low Copy (CAM)
    4. CAM control plasmid
    5. PUC19 control plasmid
  • Electroporated competent cells (α-select) with plasmids below using bioline protocol
    1. Upps 4-Kill gene-High Copy (TET)
    2. Upps 4-Upps 5-High Copy (TET)
    3. Upps 4-Upps 6-High Copy (TET)
    4. PUC19 control plasmid
    5. High copy TET control plasmid

Note 1: TET control sparked

Note 2: Upps 4-Upps 5-TET sparked

Note 3: Original DNA for Upps 4-Upps 5-TET was pink

  • Placed transformed samples in growth media and placed in 37°C shaker
  • Made 60ml of 1% agar gel for running gel electrophoresis (2 rows of 12 wells each noted below for gel) to check digestion and ligation
    • First row
      1. 2 log ladder
      2. Upps 4
      3. blank
      4. High Copy TET plasmid
      5. Upps 4 (2)
      6. Upps 6
      7. High Copy TET plasmid (2)
      8. Upps 4 (3)
      9. Kill gene
      10. High Copy TET plasmid (3)
      11. PCB
      12. High Copy TET plasmid (4)
    • Second row
      1. 2 log ladder
      2. Inducible promoter-IPTG
      3. Kill gene (2)
      4. High Copy TET plasmid (5)
      5. Inducible promoter-IPTG (2)
      6. Upps 6 (2)
      7. High Copy TET plasmid (6)
      8. Inducible promoter-IPTG (3)
      9. Upps 5 (2)
      10. High Copy TET plasmid (7)
  • Ran gel for 25 minutes at constant 150V
  • Took picture under UV light
  • Created TET and CAM plates

Wednesday, 15th

  • Diluted bacteria cultures with following plasmids to 200x LB/CAM and placed in 37°C shaker
    1. IPTG-Upps 5-Low Copy (CAM)
    2. IPTG-Upps 6-Low Copy (CAM)
    3. IPTG-Kill gene-Low Copy (CAM)
  • Purified above plasmids and CAM control plasmid using standard purification protocol and prepared glycerol stocks
  • Added appropriate buffers to IPTG-Upps 5 and IPTG-Upps 6 and centrifuged for 10 minutes to determine for a pellet
  • Measured OD 600 of following samples
    1. IPTG-Upps 5-Low Copy (CAM): .160A
    2. IPTG-Upps 6-Low Copy (CAM): .038A
  • Inserted 1µl of 1M IPTG into cultures
  • Placed all samples in 37°C overnight

Thursday, 16th

  • Measured OD 600 for 200x diluted bacterial solution containing plasmids with promoter inducible with IPTG
    1. IPTG-Upps 5-Low Copy (CAM): .040A
    2. IPTG-Upps 6-Low Copy (CAM): .032A
    3. IPTG-Kill gene-Low Copy (CAM): .028A
  • Noted that cell concentration was dense, decided to dilute solution with 75µl cells and 925µl LB/CAM solution
  • Placed diluted cell solution into 37°C incubator for 40 minutes
  • Inserted 1µl of 1M IPTG into cultures
  • Placed all samples in 37°C overnight