Team:British Columbia/Notebook

Naringenin Notebook

We kindly received the following from UBC iGEM distribution kit of 2018:
Constitutive promoter + RBS BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
4CL BBa_K801093 ~1700 BP Spring 2018 Distribution Plate 2, Well 17D pSB1C3 (Cm)
RBS+TAL BBa_K1033000 ~1600 Spring 2018 Distribution Plate 4, Well 6C pSB1C3 (Cm)


First, TAL and 4CL with RBS (ribosomal binding site) to combine them:

  • 4CL digest with EcoRI + SpeI
  • TAL digest with EcoRI + Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate and incubate at 37 C° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37*C
  • Plasmid obtained

Second, we will take bio bricks of CHS + CHI with RBS (ribosomal binding site):

  • CHS digest with EcoRI and SpeI
  • CHI digest with EcoRI and Xbal
  • Ligation of both digestion with T4 ligase
  • E. coli transformation via heat shock method
  • Spread on CMB-LB agar plate at 37c° overnight
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
  • Plasmid obtained

Third, combine the two plasmids previously prepared:

  • 4CL – TAL digest by EcoRI and SpeI
  • CHS-CHI with EcoRI and Xbal
  • Dephosphorylate CHS-CHI
  • Ligate the two together with T4 ligation
  • Add GFP via digestion and then ligation*
  • E. coli transformation via heat shock method
  • PCR and gel electrophoresis to analyze if successful
  • Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°

Protocol for cloning promoter-RBS-4CL in pSB1C3

A. Restriction enzyme digest 1. Prepare the master mix in a microfuge tube by combining the following: Component Volume to add (ul) 10X NEB Buffer 2.1 6 NEB PstI restriction enzyme 4 Autoclaved distilled water 26 TOTAL 36 2. Zip-spin the master mix to collect all liquid at the bottom of the tube 3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix 4. Vortex the master mix thoroughly 5. Zip-spin the master mix again 6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix) 7. Label the three tubes containing the aliquots as follows: a. P-RBS b. 4CL c. PDC (positive digest control)

Biosensor Notebook

July

  • 26:
    • Transformed RFP + RBS into DH5a
    • Transformed GFP + RBS into DH5a
  • 27:
    • Inoculated RFP + RBS in 25mg/uL of CM-25
    • Inoculated GFP + RBS in 25mg/uL of CM-25
  • 28:
    • Miniprepped RFP + RBS
    • Miniprepped GFP + RBS
    • Digested RFP + RBS with ECOR1 and XBA1 to obtain 8.96uL of DNA at 111.16ng/uL
    • Digested GFP + RBS with ECOR1 and XBA1 to obtain 12.8uL of DNA at 78.1ng/uL
    • Eventually discarded both digests as we did not use them

August

  • 7:
    • Transformed Bba_E0020 into DH5a
  • 8:
    • Inoculated CFP strains with 25mg/uL of CM-25
  • 9:
    • Digested CFP with ECOR1 + XBA1 to obtain 8.77uL of DNA at 114.0ng/uL
  • 15:
    • Transformed RFP into DH5a
  • 16:
    • Inoculated RFP in 25mg/uL of CM-25
    • Miniprepped and digested RFP with ECOR1 + XBA1 to obtain 9.14uL of DNA at 109.4ng/uL
  • 18:
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transformed Bba_l15016 (CFP + RBS) into DH5a
    • Synthesized parts from IDT were resuspended in 1X TBE
      • FdeR
      • Pt181-repressor
      • Pt181-GP2
      • GP2
      • Antisense
    • RFP, RFP+RBS, CFP, GFP+RBS have all been digested
  • 19:
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transfroemd CFP + RBS into DH5a
    • Both transformations were unsuccessful
  • 20
    • OD of DH5a competent cells was 0.69
    • Pelleted competent cells using 0.1M CaCl2
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transformed Bba_I5016 (CFP + RBS) into DH5a
  • 21:
    • OD of DH5a competent cells was 0.53
    • Pelleted competent cells using 0.1M CaCl2
    • Positive and Negative controls did not function properly
    • Realized glycerol was not added
    • Re-setup experiment
  • 23:
    • Reaction cleanup using ABM kit, involving:
      • GP2
      • E+S
      • Pt181-GP2
      • FdeR
    • Transformed Bba_E0040 (GFP) into DH5a
    • Transformed Bba_I15016 (CFP + RBS) into DH5a
    • Both transformations were unsuccessful
  • 24
    • Ran plasmid PSB1C3 digested with X+P, with 1X TAE buffer
    • Ran plasmid PSB1C3 digested with E+S, with 1X TAE buffer
    • Ran plasmid PSB1C3 digested with E+P, with 1X TAE buffer
    • Purified with ABM kit:
      • X+P
      • E+P
      • E+S
      • CFP
      • GFP+RBS
      • RFP+RBS
      • RFP
    • Inoculated CFP and GFP in 25mg/uL of CM-25
  • 25:
    • Miniprepped CFP+RBS and GFP (concentration 149ng/uL)
  • 27:
    • Ligated FdeR and Repressor DNA into RFP vector
    • Ligated FdER and Repressor DNA into E+S vector
    • Ligated pt181-GP2 and GP2 DNA into E+P
    • Ligated antisense DNA into X+P
  • 29:
    • Transformed in DH5a: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 30:
    • Inoculated in 25mg/uL of CM25: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 31:
    • Miniprepped: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense

Spetember

  • 4:
    • Digested: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 5:
    • Ran Gel Electrophoresis In 1X TBE buffer on: RFP - FdeR, RFP - Repressor, E+S - FdeR, E+S - Repressor, CFP - Repressor, CFP - FdeR, E+P - Pt181-GP2, E+P - GP2, X+P - Antisense
  • 7:
    • Double checked digestions with BamH1 + ECOR1 + Pst1 of strains: RFP - FdeR, E+S-FdeR
  • 13:
    • Transformed RFP - FdeR2 into DHd5a
    • Digested GFP Vector using: E+P, E+X, E+S
  • 20:
    • Ran Gel Electrophoresis on: GFP Vector + (E+P, E+X, E+S)
  • 22:
    • Purified: GFP - E+P, GFP - E+S
    • Digested Repressor (from IDT) using E+S
    • Ligated repressor with E+S vector, pt181 GP2 and GP2 with E+P digested vector. Antisense ligated with X+P vector
  • 23:
    • Inoculated: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 24:
    • Miniprepped: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 24:
    • Digested with E+P: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig
  • 25:
    • Performed Gel Electrophoresis of DNA strains: E+S - Rep Lig 1, E+P - pt181GP2 Lig, E+P - GP2 Lig, Antisense - X+P Lig

    • Kaempferol Notebook

    We kindly received the following from UBC iGEM distribution kit of 2018: '''bold'''
    Constitutive promoter + RBS BBa_K880005 ~70 BP Spring 2018 Distribution Plate 2, Well 3F pSB1C3 (Cm)
    F3H BBa_K1497009 ~1107 BP Spring 2018 Distribution Plate 2, Well 12I pSB1C3 (Cm)
    RBS BBa_B0034 ~12 BP Spring 2018 Distribution Plate 4, Well 1N pSB1C3 (Cm)
    Double Terminator BBa_B0015 ~129 BP Spring 2018 Distribution Plate 3, Well 3F pSB1C3 (Cm)
    Parts we've synthesized ourselves: '''bold'''
    FLS1 BBa_K2700000 ~1011 BP pSB1C3 (Cm)


    First, combining F3H and B0034:

    • Transform from distribution kit wells via heat shock method
    • Spread on CMB-LB agar plate at 37c° overnight
    • Pick and inoculate with LB+Cm 37c° for 16 hours
    • Miniprep to obtain plasmids (elution buffer, 50μm)
    • Digest F3H with XbaI + SpeI and B0034 with EcoRI and XbaI
    • Gel electrophoresis and gel purify
    • Ligate F3H and B0034 together with T4 ligase at room temperature for 1 hour
    • Transform via heat shock method
    • Spread on CMB-LB agar plate at 37c° overnight
    • Pick and inoculate with LB+Cm 37c° for 16 hours
    • Miniprep to obtain plasmids (elution buffer, 50μm)
    • Plasmid obtained

    Second, the FLS1 and:

    • CHS digest with EcoRI and SpeI
    • CHI digest with EcoRI and Xbal
    • Ligation of both digestion with T4 ligase
    • E. coli transformation via heat shock method
    • Spread on CMB-LB agar plate at 37c° overnight
    • PCR and gel electrophoresis to analyze if successful
    • Take positive colonies and place in LB with CMP incubate for 16 hours at 37 C°
    • Plasmid obtained

    Third, combine the two plasmids previously prepared:

    • 4CL – TAL digest by EcoRI and SpeI
    • CHS-CHI with EcoRI and Xbal
    • Dephosphorylate CHS-CHI
    • Ligate the two together with T4 ligation
    • Add GFP via digestion and then ligation*
    • E. coli transformation via heat shock method
    • PCR and gel electrophoresis to analyze if successful
    • Take positive colonies and place in LB and CMP incubate for 16 hours at 37 C°

    Protocol for cloning promoter-RBS-4CL in pSB1C3

    A. Restriction enzyme digest 1. Prepare the master mix in a microfuge tube by combining the following: Component Volume to add (ul) 10X NEB Buffer 2.1 6 NEB PstI restriction enzyme 4 Autoclaved distilled water 26 TOTAL 36 2. Zip-spin the master mix to collect all liquid at the bottom of the tube 3. With a pipette set to 25 ul, pipette each reaction up and down several times to mix 4. Vortex the master mix thoroughly 5. Zip-spin the master mix again 6. Aliquot the master mix into 3 aliquots of 14 ul (discard the remaining master mix) 7. Label the three tubes containing the aliquots as follows: a. P-RBS b. 4CL c. PDC (positive digest control)

    Plasmid Maintenance Notebook