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

September

  • 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:
    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:
    FLS1 BBa_K2700000 ~1011 BP pSB1C3 (Cm)

    July

    • 25:
      • Transformed F3H into DH5a
      • Transformed K880005 into DH5a
      • Transformed B0034 into DH5a
      • Transformed B0015 into DH5a
    • 26:
      • Inoculated F3H in 25mg/uL of CM-25
      • Inoculated K880005 in 25mg/uL of CM-25
      • Inoculated B0034 in 25mg/uL of CM-25
      • Inoculated B0015 in 25mg/uL of CM-25
    • 27:
      • Miniprepped F3H
      • Miniprepped K880005
      • Miniprepped B0034
      • Miniprepped B0015
  • 28:
    • Digested F3H with XbaI and SpeI
    • Digested K880005 with SpeI and Pst1
    • Digested B0034 with EcoR1 and Xba1
    • Digested B0015 with EcoR1 and Xba1

    • August

    • 2:
      • Ran gel and purified all samples
      • Ligate F3H (insert, 5uL) and K880005 (vector, 2uL) with T4 ligase
    • 7:
      • transformed F3H and K880005
      • Digested RFP in pSB1C3; ran gel and purified
      • Digested synthesized FLS1 with Ecor1 and Pst1
    • 10:
      • inoculated F3H and K880005
    • 15:
      • Miniprepped F3H + K880005
      • Gel ran and get purified for RFP/pSB1C3, F3H + K880005, B0034, B0015
      • gel ran and PCR purified for FLS1
    • 16:
      • Redigested F3H with XbaI and SpeI (gel failed on the 15th, needed to redo cloning)
      • Redigested K880005 with SpeI and Pst1
    • 17:
      • Ligated RFP/pSB1C3 (vector, 2uL) with FLS1 (insert, 4uL)
      • Ligated FLS1 (vector, 2uL) with B0015 (insert, 4uL)
      • purified and religated F3H+K880005
    • 19:
      • Transformed FLS1 + pSB1C3 into DH5a
      • Transformed FLS1 + B0015 into DH5a
      • Transformed F3H + K880005 into DH5a
    • 20
      • Inoculate all samples
    • 21:
      • Miniprep all samples
    • 24
      • Ran diagnostic gel for all samples, F3H + K880005 failed again, everything else fine
    • 25:
      • Took originally miniprepped F3H and digested with XbaI and SpeI
    • 27:
      • Gel purified F3H
      • ligated with previously digested B0034

      • September

    • 5:
      • Transformed F3H + B0034
    • 5:
      • Inoculated F3H + B0034
    • 7:
      • Miniprepped F3H + B0034
    • 10:
      • Ran F3H + B0034 on gel and gel purified (cloning successful!)
    • 15:
      • Used glycerol stock K880005 and streaked on LB+Cm plate
    • 16:
      • Inoculated K880005
    • 17:
      • Miniprepped K880005
    • 19:
      • Digested K880005 with Spe1 + Pst1
    • 20:
      • Ran K880005 in gel and gel purified
    • 23:
      • Digested FLS1 + B0015 with Pst1 + Spe1
      • Digested F3H + B0034 with Xba1 + Pst1
    • 24:
      • Gel eletrophoresis and gel purified FLS1 + B0015
      • Gel eletrophoresis and gel purified F3H + B0034
    • 29:
      • Ligation of F3H + B0034 (insert, 6uL) and K880005 (vector, 2uL)

      • October

    • 1:
      • Transformation of k880005 + F3H + B0034
    • 2:
      • Inoculation of k880005 + F3H + B0034
    • 3:
      • Miniprep of k880005 + F3H + B0034
    • 4:
      • Digest of k880005 + F3H + B0034

    Plasmid Maintenance Notebook