Team:Columbia-Cooper-NYC/Columbia notebook 2

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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

Tuesday, 7th

  • Prepared glycerol stock and isolated the plasmid using standard plasmid isolation protocol for following:
    1. fphA from Germany
    2. Upps 1 (pSB1K3-B0034-YF1-B0034-FixJ)
  • Prepared digestion by measuring OD at 260nm for following:
    1. Upps 1
    2. Upps 4
    3. Upps 5
    4. Upps 6
    5. Kill Gene
    6. Inducible promoter containing plasmid
  • Prepared digestions and ligated with following setup using the 3A assembly protocol provided from Bioline:
    1. Upstream: Upps 4; Downstream: Upps 5; Destination Plasmid: High Copy TET plasmid
    2. Upstream: Upps 4; Downstream: Upps 6; Destination Plasmid: High Copy TET plasmid
    3. Upstream: Upps 4; Downstream: Kill gene; Destination Plasmid: Low Copy CAM plasmid
    4. Upstream: Inducible promoter; Downstream: Upps 5; Destination Plasmid: High Copy TET plasmid
    5. Upstream: Inducible promoter; Downstream: Upps 6; Destination Plasmid: High Copy TET plasmid
    6. Upstream: Inducible promoter; Downstream: Kill gene; Destination Plasmid: High Copy TET plasmid

Wednesday, 8th

  • Pulled colony from GFP streak
  • Isolated following using standard plasmid isolation protocol:
    1. Low Copy CAM plasmid
    2. High Copy TET plasmid
    3. Inducible promoter
  • Checked optical density and applied necessary dilutions for GFP
  • Prepared two samples of GFP: sample with IPTG and sample without IPTG
  • Place samples in shaker to grow overnight

Thursday, 9th

  • Diluted GFP solutions to match proper optical density
  • Pulled colony for ho1 as backup and place in 37C inbucator

Friday, 10th

  • Remade 200 mL each of antibiotic solutions for CAM, TET, Kan, Amp
  • Conducted digestions and ligations using the 3A assembly method following the protocols provided by bioline of following:
    1. Upstream: Upps 4; Downstream: Kill gene; Destination Plasmid: High Copy TET plasmid
    2. Upstream: Inducible promoter; Downstream: Upps 5; Destination Plasmid: Low Copy CAM plasmid
    3. Upstream: Inducible promoter; Downstream: Upps 6; Destination Plasmid: Low Copy CAM plasmid
    4. Upstream: Inducible promoter; Downstream; Kill gene; Destination Plamid: Low Copy CAM plasmid
  • Followed the butanol purification protocol for ligated material containing inducible promoter from 7th and 10th

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

Tuesday, 14th

Note: Work done below was conducted at the Kanbar Lab at the Cooper Union

  • Prepared CAM from .250g of 25mg/mL CAM powder with 10mL of EtOH
  • Prepared LB/glucose media
  • Heat shocked DH5α competent cells with Inducible promoter/GFP/Low Copy CAM plasmid

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

Note: conducted the below procedures at the Kanbar Lab

  • Observed no growth for inducible promoter/GFP/low copy CAM plasmid

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

Friday, 17th

Note: IPTG induced I-U5 and I-U6 appear to give no color change (no expression)

  • Electroporate the following genes:
    1. Upps 4-Upps 6-High copy TET (sparked first time, redid trial)
    2. Upps 4-Kill-High copy TET
    3. Upps 4-Kill-Low copy CAM
    4. pUC19 control DNA (AMP resistance)
  • Placed all samples in 37°C overnight

Note2: Only adding 25µl of competent cells instead of 50µl mentioned in the

Saturday, 18th

  • Observed none of the electroporated DNA grew, but observed colonies for control
  • Placed all samples back in the 37°C incubator

Monday, 20th

  • Measured the optical density for the following:
    1. Upps 6 (with inducible promoter)
    2. Kill gene
    3. GFP
    4. Upps 5 (with inducible promoter)
  • Diluted the concentrated primer into a primer stock that would be used for DNA sequencing
  • Sent following DNA for sequencing (at Genewiz)
    1. Inducible promoter-GFP-Low Copy CAM plasmid
    2. Inducible promoter-Upps 5-Low Copy CAM plasmid
    3. Inducible promoter-Upps 6-Low Copy CAM plasmid
    4. Inducible promoter-Kill gene-Low Copy CAM plasmid
  • Analyzed gel and concluded following:
    1. The digestion for TET high copy plasmid did not work
    2. The digestion for Upps 4, 5, 6 appeared to have worked
    3. All other digestions are inconclusive results

Tuesday, 21st

  • Made additional Amp/CAM and Amp/TET plates
  • Measured the optical density for the following:
    1. Upps 1
    2. Kill gene
    3. High copy TET plasmid
  • Followed the standard digestion protocol provided by bioline or the following:
    1. Upps 1 (Upstream)
    2. Kill gene (Downstream)
    3. High copy TET plasmid (Destination plasmid)
  • Re-organized iGEM boxes by throwing out previous digestions of High Copy TET plasmids
  • Re-hydrated following biobricks from standard rehydration protocol:
    1. Constitutive promoter-BBa_J130002 (P1, 13B)
    2. Low-copy TET plasmid-PSB3T5 (P1, 7C)
    3. High-copy CAM plasmid-PSB1C3 (P1, 3A)
    4. High-copy Amp/CAM plasmid-PSB1AC3 (P1, 9A)
    5. High-copy Amp/TET plasmid-PSB1AT3 (P1, 13A)
  • 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. old TET high copy plasmid
      3. old Kill gene
      4. Upps 5
      5. IPTG inducible promoter
    • Second row
      1. 2 log ladder
      2. newly digested TET high copy plasmid
      3. newly digested Kill gene
      4. Upps 1
  • Ran gel for 25 minutes at constant 150V
  • Took picture under UV light

Wednesday, 22nd

  • Conducted ligations for the following using a vector to insert ration of 1:3
    1. Sample 1: Inducible promoter (upstream insert)-Kill gene (Downstream insert)-pSB3C5 (vector)
    2. Sample 2: Inducible promoter (upstream insert)-Upps 5 (Downstream insert)-pSB3C5 (vector)
    3. Sample 3: Inducible promoter (upstream insert)-Upps 6 (Downstream insert)-pSB3C5 (vector)
    4. Sample 4: Upps 4 (upstream insert)-Kill gene (Downstream insert)-pSB1T3 (vector)
    5. Sample 5: Upps 4 (upstream insert)-Upps 5 (Downstream insert)-pSB1T3 (vector)
    6. Sample 6: Upps 4 (upstream insert)-Upps 6 (Downstream insert)-pSB1T3 (vector)
    7. Sample 7: Control pSB3C5 (1:0 vector to insert ratio)
    8. Sample 8: Control pSB1T3 (1:0 vector to insert ratio)

Note: above ligations will be done with T4 buffer and ligase from both the Biobrick kit and Columbia's lab Note 2: label all sample using iGEM ligase and buffer with prefix N Note 3: label all sample using Columbia lab's ligase and buffer with B

  • Electroporated the following using the standard protocol provided by Bioline:
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