Team:TU Darmstadt/labbook/DetectionConstruct

From 2013.igem.org







Lab book | Materials | Protocols




Construction of the Detection Construct



The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.



Traditional cloning approach



We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.

gBLOCK approach



We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.

InFusion approach



13.08 gBLOCK assembly of CMK and TLO
  • PCR mixture
    • 1 µL of each gBLOCK
      • CMK: B_01 - B_04
      • TLO: A_01 - A_06
    • 10 µL 5x Q5 Reaction Buffer
    • 2 µL dNTPs
    • 1 µL Q5 Hot Start Polymerase
    • 10 µL 5x Q5 High GC Enhancer
    • 1 µL primer suffix-R (10 mM)
    • 1 µL primer prefix_R (10 mM)

  • PCR program (40 cycles)
    • initial denaturation 94°C, 100s
    • denaturation 94°C, 55s
    • annealing 64°C, 55s
    • elongation 72°C, 120s
    • final elongation 72°C, 300s

  • preparative 1% agarose gel
    • gel displays bands of expected size, therefore assembly was successful
13.08 Purification
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • TLO c = 34,4 ng/µL
    • CMK c = 35,6 ng/µL
19.08 Overlap extension PCR
  • reaction mixture (50 µL total volume)
    • 25 µL Q5 High Fidelity 2x Master Mix (NEB)
    • 1 µL template
    • 1 µL forward primer (10 mM)
    • 1 µL reverse primer (10 mM)
    • 22 µL nuclease-free H2O

  • template and primer specifications
    • pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev
    • CMK: CMK gBLOCK assembly + frag2_for + frag2_rev
    • terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev
    • pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev
    • TLO: TLO gBLOCK assembly + frag5_for + frag5_rev
    • pSB1C3: pSB1C3[fsC] + vector_for + vector_rev

  • PCR program (35 cycles)
    • initial denaturation: 98°C, 60s
    • denaturation: 98°C, 45s
    • annealing: 60°C, 40s
    • elongation: 72°C, 90s
    • final elongation: 72°C, 300s

  • preparative 1% agarose gel
    • gel displays byproducts
      • subsequent purification and amplification of desired bands (framed)
 x
19.08 Purification
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • pBAD1 c = 10 ng/µL
    • CMK c = 30 ng/µL
    • terminator c = 7 ng/µL
    • pBAD4 c = 11 ng/µL
    • TLO c = 22 ng/µL
    • pSB1C3 c = 16 ng/µL
19.08 Amplification PCR of pBAD1, terminator and pBAD4
  • reaction mixture (25 µL total volume)
    • 12,5 µL Q5 High Fidelity 2x Master Mix (NEB)
    • 1 µL template
    • 1 µL forward primer (10 mM)
    • 1 µL reverse primer (10 mM)
    • 9,5 µL nuclease-free H2O

  • template and primer specifications
    • pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev
    • terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev
    • pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev

  • PCR program (35 cycles)
    • initial denaturation: 98°C, 60s
    • denaturation: 98°C, 45s
    • annealing: 70°C, 30s
    • elongation: 72°C, 30s
    • final elongation: 72°C, 300s

  • analytical 1% agarose gel
    • pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature
    • terminator and pBAD4 show no bands
19.08 Gradient PCR of the amplification of pBAD1, terminator and pBAD4
  • reaction mixture (25 µL total volume)
    • 12,5 µL Q5 High Fidelity 2x Master Mix (NEB)
    • 4 µL template
    • 1 µL forward primer (10 mM)
    • 1 µL reverse primer (10 mM)
    • 6,5 µL nuclease-free H2O

  • template and primer specifications
    • pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev
    • terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev
    • pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev

  • PCR program (35 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 45s
    • annealing 55°C/57°C/61°C/65°C, 30s
    • elongation 72°C, 30s
    • final elongation 72°C, 300s

  • analytical 1% agarose gel
    • optimal annealing temperature for terminator is 55°C
    • optimal annealing temperature for pBAD4 is 55°C
 x
20.08 Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • pBAD1 c = 45 ng/µL
    • terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73
    • pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67
21.08 Amplification PCR of CMK, TLO and pSB1C3
  • PCR mixture (50 µL total volume)
    • 25 µL Q5 High Fidelity 2x Master Mix
    • 1 µL template
    • 1 µL forward primer
    • 1 µL reverse primer
    • 22 µL nuclease-free H2O

  • template and primer specifications
    • CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev
    • TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev
    • pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev

  • PCR program (35 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 45s
    • annealing 55°C, 40s
    • elongation 72°C, 90s
    • final elongation 72°C, 300s

  • analytical 1% agarose gel
    • gel displays critical amount of byproducts
22.08 Purification of CMK, TLO and pSB1C3 from preparative gel
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88
    • TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60
    • pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64
23.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 2 µL TLO (c = 37,9 ng/µL, 22.08)
    • 10 µL 5x Phusion-Buffer
    • 2 µL dNTPs
    • 2 µL DMSO
    • 4 µL Pfu-Polymerase
    • 2 µL MgCl2 (50 mM)
    • 1 µL frag5_for (10 mM)
    • 1 µL frag5_rev (10 mM)
    • 26 µL nuclease-free H2O

  • PCR program (30 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 55°C, 30s
    • elongation 72°C, 300s
    • final elongation 72°C, 600s

  • preparative 1% agarose gel
    • gel displays no distinctive bands
 x
24.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 1 µL TLO (c = 37,9 ng/µL, 22.08)
    • 10 µL 5x Phusion-Buffer
    • 2 µL dNTPs
    • 2 µL DMSO
    • 4 µL Pfu-Polymerase
    • 2 µL MgCl2 (50 mM)
    • 1 µL frag5_for (10 mM)
    • 1 µL frag5_rev (10 mM)
    • 27 µL nuclease-free H2O

  • PCR program (30 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 56°C, 30s
    • elongation 72°C, 720s
    • final elongation 72°C, 600s

  • preparative 1% agarose gel
    • gel displays no distinctive bands (just like on 23.08)
      • probably the template is poorly
24.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
    • 10 µL 5x Phusion-Buffer
    • 2 µL dNTPs
    • 2 µL DMSO
    • 4 µL Pfu-Polymerase
    • 2 µL MgCl2 (50 mM)
    • 1 µL frag5_for (10 mM)
    • 1 µL frag5_rev (10 mM)
    • 27 µL nuclease-free H2O

  • PCR program (30 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 56°C, 30s
    • elongation 72°C, 720s
    • final elongation 72°C, 600s

  • preparative 1% agarose gel
    • gel displays no distinctive bands
 x
25.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
    • 10 µL 5x Phusion-Buffer
    • 2 µL dNTPs
    • 2 µL DMSO
    • 4 µL Pfu-Polymerase
    • 2 µL MgCl2 (50 mM)
    • 1 µL frag5_for (10 mM)
    • 1 µL frag5_rev (10 mM)
    • 26 µL nuclease-free H2O

  • PCR program (30 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 55°C, 30s
    • elongation 72°C, 300s
    • final elongation 72°C, 600s

  • preparative 1% agarose gel
    • gel displays the desired band of 2500 bp as well as byproducts
25.08 Purification of TLO
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • TLO c = 19 ng/µL
25.08 InFusion
  • reaction mixture (71 µL total volume)
    • 1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
    • 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
    • 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
    • 42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)
    • 4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
    • 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
    • 18µl 5x InFusion Pfu MasterMix

  • treatment of the reaction mixture according to InFusion protocol


  • transformation into E.coli Top10 according to heat shock protocol
    • no colonies grew on LB-Cam plates, most likely the reaction volume was to high
26.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)
    • 1 µL frag5_rev (10 mM)
    • 1 µL frag5_for (10 mM)
    • 10 µL 5x Phusion Buffer
    • 27 µL nucleasefree H2O
    • 2 µL dNTPs
    • 2 µL DMSO
    • 2 µL MgCl2 (50 mM)
    • 4 µL Pfu-Polymerase

  • PCR program (30 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 55°C, 30s
    • elongation 72°C, 300s
    • final elongation 72°C, 600s

  • preparative 1% agarose gel
    • gel displays the desired band of 2500 bp as well as byproducts
 x
26.08 Purification of TLO
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • TLO c = 19,7 ng/µL
26.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 1 µL TLO (c = 19,7 ng/µL)
    • 1 µL frag5_rev (10 mM)
    • 1 µL frag5_for (10 mM)
    • 10 µL 5x Phusion Buffer
    • 27 µL nucleasefree H2O
    • 2 µL dNTPs
    • 2 µL DMSO
    • 2 µL MgCl2 (50 mM)
    • 4 µL Pfu-Polymerase

  • PCR program (35 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 55°C, 30s
    • elongation 72°C, 300s
    • final elongation 72°C, 600s

  • preparative 1% agarose gel
    • no distinctive bands
 x
27.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)
    • 1 µL frag5_rev (10 mM)
    • 1 µL frag5_for (1:10)
    • 10 µL 5x Phusion Buffer
    • 25,5 µL nucleasefree H2O
    • 2 µL dNTPs (10 mM)
    • 2,5 µL DMSO
    • 2 µL MgCl2 (50 mM)
    • 4 µL Pfu-Polymerase
    • 2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)

  • PCR program (35 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 55°C, 30s
    • elongation 72°C, 480s
    • final elongation 72°C, 600s

  • preparative 1% agarose gel
    • no distinctive bands (just like on 26.08)
28.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)
    • 2,5 µL primer frag5_rev (10 mM)
    • 2,5 µL primer frag5_for (10 mM)
    • 19 µL nucleasefree H2O
    • 25 µL 2x Q5 Mastermix (NEB)

  • PCR program (35 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 66°C, 30s
    • elongation 72°C, 90s
    • final elongation 72°C, 120s

  • preparative 1% agarose gel
    • no distinctive bands (just like on 26.08)
      • probably the template is poorly
28.08 Amplification of TLO
  • PCR mixture (50 µL total volume)
    • 1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)
    • 2,5 µL primer frag5_rev (10 mM)
    • 2,5 µL primer frag5_for (10 mM)
    • 19 µL nucleasefree H2O
    • 25 µL 2x Q5 Mastermix (NEB)

  • PCR program (35 cycles)
    • initial denaturation 98°C, 60s
    • denaturation 98°C, 30s
    • annealing 66°C, 30s
    • elongation 72°C, 90s
    • final elongation 72°C, 120s

  • preparative 1% agarose gel
    • gel displays the desired band of 2500 bp as well as byproducts
 x
29.08 Purification of TLO
  • using Wizard SV Gel and PCR Clean-Up System (Promega)
    • TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02
31.08 InFusion
  • reaction mixture (20 µL total volume)
    • 1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)
    • 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)
    • 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)
    • 3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)
    • 4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)
    • 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)
    • 4µl 5x InFusion Pfu MasterMix
    • 1,5µl nucleasefree H2O

  • treatment of the reaction mixture according to InFusion protocol


  • transformation into E.coli Top10 according to heat shock protocol
    • 2 colonies grew on LB-Cam plates, most likely the reaction volume was to high
02.09 Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction
  • Colony PCR
    • PCR mixture (50 µL total volume)
      • 1µl VR primer (10 mM)
      • 1µl VF2 primer (10 mM)
      • 5µl Colony LB Medium (Colony 1/Colony 2)
      • 2µl MgCl2
      • 1µl dNTP Mix
      • 1µl DMSO
      • 5µl 10x Taq Buffer
      • 1µl Taq-Polymerase
      • 33µl nucleasefree H2O

    • PCR Programm
      • initial denaturation 300s 95°C
      • denaturation 30s 95°C
      • annealing 30s 55°C
      • elongation 150s 72°C
      • final elongation 300s 72°C

  • Purification using Pure Yield Plasmid Miniprep System (Promega)
    • Colonie 1 = pSB1C3-TLO-CMK 1
      • c = 245,5 ng/ul
      • 260/280 = 1,87
      • 260/230 = 2,23

    • Colonie 2 = pSB1C3-TLO-CMK 2
      • c = 107,7 ng/ul
      • 260/280 = 1,95
      • 260/230 = 2,28

  • digestion with EcoRI and double digest with EcoRI and PstI

  • analytical 1% agarose gel
    • pattern is as expected
    • colony PCR product is off by 2000 bp
    • linearized plasmid is off by 1000 bp
 x
03.09 Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR
  • PCR mixture (50 µL total volume)
    • 1µl forward primer
    • 1µl reverse primer
    • 1µl template
    • 2µl MgCl2
    • 1µl dNTP Mix
    • 1µl DMSO
    • 5µl 10x Taq-Buffer
    • 1µl Taq-Polymerase
    • 37µl H2O

  • template and primer specifications
    • template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution
      • PCR tube 1: frag1_for + frag4_rev
      • PCR tube 2: frag2_for + frag2_rev
      • PCR tube 3: frag3_for + frag3_rev
      • PCR tube 4: frag4_for + frag4_rev
      • PCR tube 5: frag5_for + frag5_rev

    • template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution
      • PCR tube 1: frag1_for + frag4_rev
      • PCR tube 2: frag2_for + frag2_rev
      • PCR tube 3: frag3_for + frag3_rev
      • PCR tube 4: frag4_for + frag4_rev
      • PCR tube 5: frag5_for + frag5_rev

  • PCR program (30 cycles)
    • initial denaturation 300s 95°C
    • denaturation 30s 95°C
    • annealing 30s 55°C
    • elongation 150s 72°C
    • final elongation 300s 72°C

  • analytical 1% agarose gel
    • besides many byproducts all fragments could be amplified from templates (framed bands)
      • clones are most likely correct
 x
03.09 Induction of pSB1C3-TLO-CMK clones with L-Arabinose
  • induction with L-arabinose (0,02% w/v) at OD600 = 0,6
    • induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer
03.09 Sequencing of pSB1C3-TLO-CMK clones
  • were not able to sequence the whole insert, as pimers did not anneal sufficiently
  • results indicate that all five fragments were successfully ligated in the correct order
  • results show that gBLOCK assembly of TLO and CMK did not work correctly

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