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From 2013.igem.org

Construction of the Detection Construct

Labbook |	Protocols |	Materials |

Contents 1 The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange 1.1 traditional cloning approach 1.2 gBLOCK approach 1.3 InFusion approach

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)
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
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
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
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
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
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
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
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
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