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 ratio 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:
    1. Constitutive promoter-BBa_J130002
    2. Low-copy TET plasmid-PSB3T5
    3. High-copy CAM plasmid-PSB1C3
    4. High-copy Amp/CAM plasmid-PSB1AC3
    5. High-copy Amp/TET plasmid-PSB1AT3
    6. PUC19 control plasmid
  • Chemically transformed the following using the heat shock protocol:
    1. N1, N2, N3, N7, B1, B2, B3, B7 into BL21 (DE) pLysZ orange cells
    2. GFP IPTG given by graduate student at Columbia into BL21 (DE) green cells
    3. PUC19 into BL21 (DE) pLysZ orange cells
    4. PUC19 into BL21 (DE) green cells
  • Purified ligated protocol for electroporation following QIAquick PCR purification protocol
  • Plated all chemically transformed and electroporated cells

Thursday, 23rd

Note: Observed no growth with pSB1T3, BL21 pLyse

Note 2: Observed growth with PUC19 and other BL21

  • Streaked sample N5
  • Found following 3 restriction sites (to be double checked by graduate student):
    1. NgomIV: CCGCCGGC
    2. EcoRI: GAATTC
    3. PstI: CTGCAG

Friday, 24th

  • Diluted IPTG driven cultures to 200x with fresh LB/antibiotic solution
  • Followed standard plasmid isolation protocol for everything except the following:
    1. 1 sample of graduate student's GFP
    2. Sample of pSB1C3 (the high copy CAM plasmid)
  • Checked optical densities of samples B1-B3 and graduate student's GFP
  • Created two subsamples each of B1-B3; applied 1M IPTG to half of the subsamples
  • Applied 1M IPTG to into one of the samples of graduate student's IPTG-GFP

Saturday, 25th

  • Checked IPTG "induced" samples and observed no expression
  • Measured optical density for following samples and diluted the samples:
    1. GFP by graduate student-pre-dilution: 2.0, post dilution: 0.710
    2. IPTG-kill (N1)-pre-dilution: 2.4, post dilution: 0.736
    3. IPTG-U5 (N2)-pre-dilution: 1.9, post dilution: 0.740
    4. IPTG-U6 (N5)-pre-dilution: 2.4, post dilution: 0.630
    5. IPTG-U5 (B2)-pre-dilution: 1.9, post dilution: 0.681
    6. IPTG-U6 (B3)-pre-dilution: 1.9, post dilution: 0.651
  • Applied 1.5μL of 1M IPTG with 1.5mL of each sample and place in 25C shaker
  • Followed standard plasmid isolation protocol for N1 and N5

Monday, 27th

  • Observe expression of graduate student's GFP
  • Measured optical densities for following:
    • Ligated samples:
      1. N1-N3
      2. B1-B3
    • Mini-prepped:
      1. Upps1-Upps6
      2. pSB1C3
      3. pSB1T3
      4. pSB1AT3
      5. pSB1AC3
      6. pSB1A2
  • Sent above samples for sequencing
  • Followed protocol from bioline for digestion:
    1. Downstream: Upps 1
    2. Upstream: Constitutive promoter
    3. Destination plasmid: pSB3T5
    4. Destination plasmid: pSB1C3
    5. Destination plasmid: pSB1AC3
    6. Destination plasmid: pSB1AT3
  • Streaked out Upps 1 from agar stab

Tuesday, 28th

  • Reviewed results from the sequencing from Genewiz
  • Design and send new primer (nev) for sequencing
  • Conducted ligations for the following using a vector to insert ratio of 1:3
    1. Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1C3 (vector)
    2. Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB3T5 (vector)
    3. Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1AT3 (vector)
    4. Constitutive promoter (upstream insert)-Upps 1 (downstream insert)-pSB1AC3 (vector)
  • Electroporated the above 4 ligations without purifying the ligations
  • Plated cells into respective plates
  • Picked colonies for Upps 1-YF1/FixJ

Wednesday, 29th

  • Conducted ligations for the following using a vector to insert ratio of 1:3
    1. Upps 4 (upstream insert)-Upps 5 (downstream insert)-pSB1C3 (vector)
    2. Upps 4 (upstream insert)-Upps 6 (downstream insert)-pSB1C3 (vector)
    3. Upps 4 (upstream insert)-Kill gene (downstream insert)-pSB1C3 (vector)
    4. Upps 4 (upstream insert)-Upps 5 (downstream insert)-pSB1AC3 (vector)
    5. Upps 4 (upstream insert)-Upps 6 (downstream insert)-pSB1AC3 (vector)
    6. Upps 4 (upstream insert)-Kill gene (downstream insert)-pSB1AC3 (vector)
  • Extracted Upps 1 (Kan) from glycerol stock from -80C freezer
  • Followed graduate student's quickchange protocol and labelled N1-N4, E1-E4, P1-P4 with the labeling as following:
    1. Sample 1: HF only
    2. Sample 2: HF and 1.5μL DMSO
    3. Sample 3: GC only
    4. Sample 4: GC and 1.5μL DMSO
    5. Prefix N: NgoMIV mutation
    6. Prefix E: EcoRI mutation
    7. Prefix P: PsH mutation

Thursday, 30th

  • Followed standard plasmid isolation protocol for Upps 1
  • Transformed U4-U5, U4-U6, U4-kill in 2 different plasmids
  • Ran gels of Quickchange product
    1. Column 1: miniprepped plasmid
    2. Column 2-5: N1-N4
    3. Column 6-9: E1-E4
    4. Column 10-13: P1-P4
  • Re-ligated Constitutive promoter-Upps 1

September, 2012

Wednesday, 5th

Note: All work done from this day on was done at the Kanbar lab at the Cooper Union

  • Single digested the J13002
  • Placed the digest above and other digests to be run on gels into -20C freezer
  • Made 4 50mL 1% agarose gels
  • Followed standard plasmid isolation protocol for kill gene

Friday, 7th

  • Ran two gels to check digests
    • Gel Number 1
      1. Marker
      2. Upps 1
      3. Inducible promoter 1
      4. Inducible promoter 2
      5. pSB3T5
      6. pSB1C3
      7. Low Copy Vector 1
      8. Low Copy Vector 2
    • Gel Number 2
      1. Marker
      2. Kill gene
      3. PcyA
      4. TetR 1
      5. TetR 2
      6. GFP from biobrick kit
      7. GFP from Tushar
      8. GFP from Kevin
  • Conducted digestions for the following:
    1. Inducible promoter digested with EcoRI&Spe with Buffer E and Buffer Multi (for 30 min.)
    2. TetR digested with EcoRI with Buffer H
    3. TetR digested with Spe with Buffer B
    4. Upps 1 digested with Xba&Pst with Buffer H
    5. Kill gene digested with Xba&Pst with Buffer H
    6. GFP digested with Xba&Pst with Buffer H
    7. pSB3T5 with EcoRI&Pst with Buffer H
    8. Low copy plasmid 2 digested with EcoRI&Pst with Buffer H
    9. pSB1C3 digested with EcoR&Pst with Buffer H

Note: Digests 7-9 was done in Antartic Phosphotase Treat 15 min in 37C and 5 min in 65C

  • Conducted ligations for the following (followed by chemical transformations:
    1. Upstream: Inducible promoter; Downstream: Upps 1; Destination plasmid: Low copy plasmid 2
    2. Upstream: Inducible promoter; Downstream: Kill; Destination plasmid: Low copy plasmid 2
    3. Upstream: Inducible promoter; Downstream: GFP; Destination plasmid: Low copy plasmid 2
  • Conducted additional chemical transformations with the following vectors:
    1. pET20b
    2. pET26b
    3. pUC19
  • Conducted following additional ligations, reference numbers correlate to numbers shown above for this day's digestions
    1. Label A: 2 and 9
    2. Label B: 3 and 4 and 9
    3. Label C: 1 and 4 and 8
    4. Label D: 1 and 5 and 8
    5. Label E: 1 and 6 and 8
    6. Label F: 3 and 4
    7. Label G: 3 and 5
    8. Label H: 3 and 6

Tuesday, 11th

  • Observed the following:
    1. Inducible promoter-GFP-Low Copy CAM Plasmid had 3 pink clones (IPTG applied)
    2. Inducible promoter-GFP-Low Copy CAM Plasmid had 3 pink clones (NO IPTG)
    3. Inducible promoter-Upps 1-Low Copy CAM Plasmid had 32 pink clones
    4. Inducible promoter-Kill gene-Low Copy CAM Plasmid had 50 pink clones

Note 1: Destination plasmid/vector contained J04450 biobrick part, explained pink clones

Note 2: All vectors were all treated with Antartic Phophotase, expect no religation of J04450 with LC vector

  • Digested Kill gene and GFP with E and P
  • AP Treat Kill gene with X and P, GFP with X and P
  • Conducted ligations for the following:
    1. GFP (E and P) with pSB1C3 (E and P, AP treated)
    2. GFP (X and D, AP treated) with TetR/s
  • Condcuted transformations for the following:
    1. Ligation F from 9/7, TetR, Upps 1(X and P)
    2. Kill gene, pSB1C3
    3. GFP, pSB1C3
    4. GFP, TetR
  • Mini-cultured pET20b and pET26b

Wednesday, 12th

  • Observed following from the transformations from 9/11
    1. TetR/GFP-successful with about 250 colonies
    2. TetR/Upps 1-successful with about 200 colonies
    3. No growth with Kill&pCB1C3, GFP&pCB1C3
  • Prepared colony PCR for TetR&GFP, and TetR&Upps 1 on Amp grid plate (had A-D columns and 1-4 rows)
    1. Resuspended single colonies in 4μL deionized water
    2. Transferred 3μL colony suspension to Amp grid
    3. Stored remaining 1μL as PCR template
    4. Grew Amp grid plate in 37C overnight

Note 1: GFP/TetR: A and B with 1-4 each

Note 2: Upps 1/TetR: C and D with 1-4 each

  • PCR reaction contained following mixture:
    • 1x PCR Rxn
    1. 1.0μL of 10x buffer
    2. 0.4μL of dNTP
    3. 0.4μL of Primer F
    4. 0.4μL of Primer R
    5. 1.0μL of template mentioned above
    6. 0.4μL of tag
    7. 6.4μL of deionized water
    • 17x PCR Rxn
    1. 17μL of 10x buffer
    2. 6.8μL of dNTP
    3. 6.8μL of Primer F
    4. 6.8μL of Primer R
    5. 6.8μL of tag
    6. 108.8μL of deionized water
  • PCR reactions conducted with following protocol:
    1. Denature at 95C for 10 min.
    2. Denature at 95C for additional 30 sec.
    3. Anneal at 56C for 30 sec.
    4. Elongation at 70C for 60 sec.
    5. Elongation at 72C for 20 min.
    6. Hold at 4C for 39 cycles
  • Set up second half composite construction digests for following:
    1. Upps 1; Enzyme: EcoRI and SpeI; Buffer E*; Ratio: 1:2; Reaction vol.: 30μL
    2. Upps 4; Enzyme: EcoRI and SpeI; Buffer E*; Ratio: 1:2; Reaction vol.: 30μL
    3. Upps 5; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
    4. Upps 6; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
    5. Kill gene; Enzyme: Xba and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
    6. Linearlized pSB1T3; Enzyme: EcoRI and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
    7. Linearlized pSB1K3; Enzyme: EcoRI and Pst; Buffer H; Ratio: 1:1; Reaction vol.: 20μL
    8. Promoterless GFP; Enzyme: Xba; Buffer H; Ratio: N/A; Reaction vol.: 20μL
    9. pUC19; Enzyme: EcoRI; Buffer H; Ratio: N/A; Reaction vol.: 20μL

Note 3: for * buffers, will use buffer 2 or 4 in future

  • Reactions took place for 1 hr. in 37C incubation and placed in -20C
  • Isolated pGLO and pET20b vectors using standard plasmid isolation protocol
  • Prepared glycerol stocks for pET20b and pET26b

Thursday, 13th

  • Conducted fphA PCR mutogenesis with following (template = pASKfphA 753bp)
    • 1x PCR Rxn
    1. 2.0μL of 10x Buffer
    2. 0.4μL of dNTP
    3. 0.4μL of Primer F (EcoRI)
    4. 0.4μL of Primer R (EcoRI)
    5. 1.0μL of template
    6. 0.4μL of tag
    7. 15.4μL of deionized water
    • 3x PCR Rxn
    1. 6.0μL of 10x Buffer
    2. 1.2μL of dNTP
    3. 1.2μL of Primer F (EcoRI)
    4. 1.2μL of Primer R (EcoRI)
    5. 1.2μL of tag
    6. 46.2μL of deionized water
  • Set up 4 min. extension time with 55C for annealing temp for PCR program
  • Digested above mixture with Dpn1 at 37C for 1hr. after PCR
  • Transformed product into DH5α
  • Heat inactivated samples with Spe1 restriction digests from 9/12 at 80C for 20 min. and rest at 65C for 20 min.
  • Antartic treated the following (15 min. 37C, 5 min. 65C):
    1. Upps 4
    2. Promoterless GFP
    3. pUC19
  • Conducted ligations for the following for a total volume of 10μL
    1. Upps 4-Kill gene-pSB1K3
    2. Upps 4-Upps 5-pSB1K3
    3. Upps 4-Upps 6-pSB1K3
    4. TetR/Spe (from 9/7)-promoterless GFP
    5. pUC19
  • Transformed ligated DNA into DH5α
  • Ran gels for PCR Rxn and observed following:
    1. A3, B3 had 300 bp (estimated) product
    2. B1, B4 had 700 bp (estimated) product
    3. Everything else (including marker failed)
    4. Can not differentiate between ±TetR
  • Minicultured 4 TetR-Upps 1 cultures randomly

Friday, 14th

  • Sterilized glycerol for stocks
  • Created more LB/Amp plates
  • Created more sterile miniculture tubes
  • Ran 100 bp optimization gel

Note 1: While 2µl DNA visble, should use minimum of 5µl/lane for system (SYBR)

  • Repeated VF2-VR PCR on TetR&promoterless GFP (denoted A) and TetR&Upps 1 (denoted B)
    • 1x PCR Rxn
      1. 1.0μL of 10x Buffer
      2. 0.4μL of dNTP
      3. 0.4μL of Primer F (EcoRI)
      4. 0.4μL of Primer R (EcoRI)
      5. 1.0μL of template
      6. 0.4μL of tag
      7. 6.4μL of deionized water
    • 17x PCR Rxn
      1. 17.0μL of 10x Buffer
      2. 6.8μL of dNTP
      3. 6.8μL of Primer F (EcoRI)
      4. 6.8μL of Primer R (EcoRI)
      5. 6.8μL of tag
      6. 108.8μL of deionized water

Note 2: Will pick 7 colonies from each and have 4 minicultures of B-will use 1µl±1 colony

  • After review of primers, changed PCR program as follows:
    1. Increased annealing temp to 60C
    2. Increased extension temp to 70C
    3. Reduced cycles to 30

Thursday, 20th

  • Reviewed order of custom primer from Invitrogen (primer ordered: 9/17)
  • Rehydrated primers
  • Ran PCR Rxn with following:
    1. Annealing temperature: 61C
    2. Elongation time: 3 minutes
    3. 30 cycles

Monday, 24th

  • Reviewed plates and observed the following:
    • Noted plenty of colonies on both plates, as expected
    • Plates with pSB1C3-J04450 based vector had many pink colonies and 8-10 white clones
    • Considering both vectors were alkaline phosphatase treated, will assume treatment failed for both vectors
    • Will currently disregard Upps 3 based vector transformations
  • Picked following colonies for miniculture:
    1. pSB1C3-FphA
    2. Upps 4-Kill gene
    3. Upps 4-Upps 6
    4. TetR-Upps 1
  • Minicultured the following from glycerol stocks
    1. Upps 1
    2. Upps 4
    3. TetR

Tuesday, 25th

  • Ran a gel to verify following plasmids:
    • Sizing Gel 1
      1. Kill gene 1 (OK)
      2. Kill gene 2 (OK)
      3. FphA1 (too small - discard)
      4. FphA2 (OK)
      5. Upps 1 (too big - discard)
      6. B0034 RBS (OK, faint)
    • Sizing Gel 2
      1. Upps 4-Kill gene 1 (OK)
      2. Upps 4-Kill gene 2 (OK)
      3. Upps 4-Upps 6 (OK)
      4. Upps 4-Upps 6 (too big - discard)
      5. Upps 4 (OK)
      6. B0030 RBS.1 (OK, but really faint)

Note 1: the parenthesis denote observations/analysis of gel

  • Isolated plasmids using standard miniprep protocol for the minicultures from previous day
  • Set up PCR site directed mutagenesis using pSB1C3-FphA as template

Note 2: used protocol from 9/13 and EcoRI primer set

  • Rehydrated the following using standard iGEM rehydration protocol
    1. BBa_J23100 (plate 1, 18C [Amp])
    2. BBa_J23101 (plate 1, 18E [Amp])
    3. Bba_J23102 (plate 1, 18G [Amp])
  • Transformed the above rehydrated biobricks into DH5α

Wednesday, 26th

  • Digested the PCR Rxn with DpnI
  • Transformed pSB1C3-FphA into DH5α
  • Digested the following:
    1. BBa_B0034 RBS with SpeI
    2. Upps4 with XbaI
    3. Kill gene with XbaI
  • Ligated the following (was predigested with EcoRI):
    1. RBS-Upps 4
    2. RBS-Kill gene
  • Gel extracted the bands of the following:
    1. RBS-Upp4 Target 2352bp, erroneous 2155bp
    2. RBS-Kill Target 3441bp, erroneous 2155bp
  • Minicultured the following:
    1. Upps 1
    2. Promoterless GFP
    3. Promoters 100-102

Wednesday, 27th

  • Made minipreps from minicultures from previous day, ran qualitative gel, and concluded the following:
    1. Upps 1 had poor yield
    2. Promoterless GFP showed no growth
  • Conducted digestions of the following: (yielded 2x volume)
    1. RBS with SpeI
    2. Promoter 102 with SpeI
    3. Upps 1 with XbaI
    4. Promoterless GFP with XbaI
  • Conducted ligations of the following using digestion with 1x volume of EcoRI: (yielded 2x volume)
    1. Promoter 102-Upps 1
    2. Promoter 102-Promoterless GFP
    3. RBS-Upps 4
    4. Ribosome Binding Site-Kill gene
  • Ran 1x volume on gel to purify and gel extract target band
  • Transformed the above in DH5α using standard heat shock protocol
  • Observed the PCR site mutagenesis yielded no colonies

9/28/2012 =

RBS+Kill worked; everything else did not grow :( Miniculture RBS-Kill x3 promoterless GFP in [Kan] Upp1 Upp4 Digest Upp1 with XbaI Inducible Promoter with SpeI Upp1 with EcoRI/SpeI Upp4 with XbaI/PstI pSB1A3 (linearized DNA) with EcoRI/PstI pSB3T5 with EcoRI/PstI Ligate Promoter 100-Upp1 Promoter 101-Upp1 Promoter 102-Upp1 Inducible Promoter-Upp1 Promoter 102-promoterless GFP Upp1-Upp4-pSB1A3 Upp1-Upp4-pSB3T5 Transform into DH5α

Saturday, 29th

  • Observed that Promoter 102-GFP worked and everythign else did not
  • Conducted digestions of the following:
    1. Upps 1 with EcoRI&SpeI
    2. Upps 4-Kill with XbaI&PstI
    3. Upps 4-Upps 6 with XbaI&PstI
    4. pSB1C3-J04450 with EcoRI&PstI
  • Conducted ligations of the following:
    1. Upps 1-Upps 4-Kill gene-pSB1C3 destination plasmid
    2. Upps 1-Upps 4-Upps6-pSB1C3 destination plasmid
  • Transformed the above ligated products into DH5α using a heat shock protocol
  • Transformed the following into JM109:
    1. Promoter 100-Upps 1
    2. Promoter 101-Upps 1
    3. Promoter 102-Upps 1
    4. Inducible Promoter-Upps 1

October, 2012

Monday, 1st

  • Observed that all of the plates grew colonies
  • Prepared minicultures of the following:
    1. Upps 1-Upps 4-Kill gene-pSB1C3 destination plasmid
    2. Upps 1-Upps 4-Upps 6-pSB1C3 destination plasmid
    3. Promoter 102-Upps 1
    4. Inducible promoter-Upps 1

Tuesday, 2nd

  • Isolated following plasmids by following standard plasmid isolation protocol and submitted parts:
    1. pSB1C3-FphA
    2. Upps 4-Kill gene
    3. Upps 1-Upps 4-Kill gene
    4. Upps 1-Upps 4-Upps 6
    5. Promoter 102-Upps 1
    6. Inducible promoter-Upps 1
Retrieved from "http://2012.igem.org/Team:Columbia-Cooper-NYC/Columbia_notebook_2"