Team:TU Darmstadt/labbook/DetectionConstruct

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Construction of the Detection Construct

Labbook | Protocols | Materials |

Contents

The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange

traditional cloning approach

was abbanndoned due to major difficulties during restriction, ligation and transformation

gBLOCK approach

was abbanndoned 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
  • 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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