Team:TU Darmstadt/labbook/Biobricks

From 2013.igem.org

Lab book | Materials | Protocols

BioBricks mKate and LssmOrange

01.08	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 ' assembly was successful
01.08	Purification
	using Wizard SV Gel and PCR Clean-Up System (Promega) TLO c = 7,7 ng/µL CMK c = 7,1 ng/µL
02.08	Isolation of LssmOrange and mKate
	PCR mixture (50 µL total volume) 1.5 µl DMSO 1 µL dNTPs 4 µL Pfu Polymerase 10 µL 5x Pfu buffer Buffer with MgSO4 1 µL reverse primer 1 µL forward primer 5 µl template add 50 µl H2O template and primer specifications LssmOrange: TLO (c = 7,7 ng/µL, 01.08) + LO-pre-F + LO-suf-R mKate: CMK (c = 7,1 ng/µL, 01.08) + mKate-suf-R + mKate-pre-ATG-F PCR program (35 cycles) initial denaturation 95°C, 300s denaturation 95°C, 30s annealing 55°C, 55s elongation 72°C, 2 min final elongation 72°C, 300s preparative 1% agarose gel PCR batches show the expected bands ' isolation was successful
02.08	Purification
	using Wizard SV Gel and PCR Clean-Up System (Promega) LssmOrange1 c = 78,5 ng/µL 260/280 = 1,87 230/260 = 1,19 mKate1 c = 45,8 ng/µL 260/280 = 1,78 230/260 = 0,49 mKate2 c = 44,9 ng/µL 260/280 = 1,89 230/260 = 0,47
02.08	Restriction of LssmOrange and mKate
	double digest with PstI and EcoRI for 1 hour at 37°C
03.08	Purification
	using Wizard SV Gel and PCR Clean-Up System (Promega) LssmOrange1 c = 51,6 ng/µL 260/280 = 1,82 230/260 = 0,63 mKate1 c = 45,4 ng/µL 260/280 = 1,9 230/260 = 0,48 mKate2 c = 17,0 ng/µL 260/280 = 1,87 230/260 = 0,12
03.08	Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate
	ligation mixture 120 ng pSB1C3 cut with EcoRI and PstI 124 ng insert cut with EcoRI and PstI 1 µL T4 DNA ligase (NEB) 2 µL 10x t4 DNA ligase buffer (NEB) add to 20 µL incubation for 30 minutes at room temperature
03.08	Transformation
	2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol only few colonies grew on LB-Cam plates
05.08	Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones
	PCR mixture (50 µL total volume) 1µl VR primer (10 mM) 1µl VF2 primer (10 mM) 5µl bacterial culture in LB-Cam medium 2µl MgCl2 1µl dNTP Mix 1µl DMSO 5µl 10x Taq Buffer 1µl Taq-Polymerase 33µl nucleasefree H2O PCR Program initial denaturation 300s 95°C denaturation 30s 95°C annealing 30s 55°C elongation 60s 72°C final elongation 300s 72°C analytical 1% agarose gel no colony yielded a band of approximately 800 bp, which would account for a positive result
09.08	Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate
	ligation mixture 120 ng pSB1C3 cut with EcoRI and PstI 124 ng insert cut with EcoRI and PstI 1 µL T4 DNA ligase (NEB) 2 µL 10x t4 DNA ligase buffer (NEB) add to 20 µL incubation for 2 hours at 37°C and additionally incubation over 24 hours at 8°C
10.08	Transformation
	2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol a lot of colonies grew on LB-Cam plates
12.08	Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones
	PCR mixture (50 µL total volume) 1µl VR primer (10 mM) 1µl VF2 primer (10 mM) 5µl bacterial culture in LB-Cam medium 2µl MgCl2 1µl dNTP Mix 1µl DMSO 5µl 10x Taq Buffer 1µl Taq-Polymerase 33µl nucleasefree H2O PCR Program initial denaturation 300s 95°C denaturation 30s 95°C annealing 30s 55°C elongation 60s 72°C final elongation 300s 72°C analytical 1% agarose gel a lot clones gave a positive result
13.08	Sequencing of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones
	results show that all analyzed clones contain the expected sequence one pSB1C3[LssmOrange] clone and one pSB1C3[mKate] clone were chosen
26.08	Plasmid prep of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones
	using PureYield Plasmid Miniprep System (Promega) pSB1C3[LssmOrange] c = 96,4 ng/µL 260/280 = 1,68 230/260 = 1,46 pSB1C3[mKate] c = 74,8 ng/µL 260/280 = 1,86 230/260 = 1,82
13.09	Inoculation of DH5? pPR-IBA2 and subsequent plasmid prep
	6 mL LB-Amp were inoculated with 50 µL of DH5? pPR-IBA2 using PureYield Plasmid Miniprep System (Promega) pPR-IBA2 c = 71,6 ng/µL 260/280 = 1,82 260/230 = 1,76
13.09	Isolation PCR of LssmOrange and mKate
	PCR mixture (50 µL total volume) 5x Q5 Reaction Buffer 5x Q5 GC Enhancer 1 µL dNTPs (10mM) 2,5 µL forward primer (10 mM) 2,5 µL reverse primer (10 mM) 1 µL template 0,5 µL Q5 Polymerase 22,5 µL nucleasefree water template and primer specifications LssmOrange: pSB1C3[LssmOrange ] (c = 96,4 ng/µL, 1:100 dilution) + lssmorange pprf + lssmorange ppr mKate: pSB1C3[mKate] (c = 74,8 ng/µL, 1:80 dilution) + mkate pprf + mkate ppr PCR program (35 cycles) initial denaturation, 60 s, 98°C denaturation, 10 s, 98°C annealing, 30 s, 65°C elongation, 60 s, 72°C final elongation, 120 s, 72°C analytical 1% agarose gel each PCR batch displays the expected band of approximately 700 bp
13.09	Purification
	using Wizard SV Gel and PCR Clean-Up System (Promega) LssmOrange1 c = 51,4 ng/µL 260/280 = 1,69 230/260 = 1,59 LssmOrange1 c = 51,6 ng/µL 260/280 = 1,66 230/260 = 1,55 mKate1 c = 38,4 ng/µL 260/280 = 1,64 230/260 = 1,43 mKate2 c = 37,8 ng/µL 260/280 = 1,59 230/260 = 1,42
13.09	Restriction of pPR-IBA2, LssmOrange and mKate
	double digest with NheI and PstI at 37°C for 4 hours preparative 1% agarose gel LssmOrange and mKate show a band of the expected size pPR-IBA2 shows intense band at 3000bp, weak band at 2250bp and weak band below 100bp we were unsure which band the correct one is, and cut out the 3000band as well as 2250band
14.09	Purification of pPR-IBA2, LssmOrange and mKate
	using Wizard SV Gel and PCR Clean-Up System (Promega) pPR-IBA2 (3000band) c = 31,2 ng/µL 260/280 = 1,88 230/260 = 1,00 pPR-IBA2 (2250band) c = 9,5 ng/µL 260/280 = 1,39 230/260 = 0,38 (neglected due to low purity) LssmOrange c = 57,3 ng/µL 260/280 = 1,74 230/260 = 1,53 mKate c = 22,0 ng/µL 260/280 = 2,04 230/260 = 1,16
14.09	Ligation
	ligation batch of pPR-IBA2[LssmOrange] 3 µL pPR-IBA2 3000 (93 ng) 1,5 µL LssmOrange (72 ng) 2 µL 10x T4 Ligase Buffer (NEB) 1 µL T4 Ligase (NEB) 12,5 µL nucleasefree water ligation batch of pPR-IBA2[mKate] 3 µL pPR-IBA2 3000 (93 ng) 3,5 µL mKate (72 ng) 2 µL 10x T4 Ligase Buffer (NEB) 1 µL T4 Ligase (NEB) 10,5 µL nucleasefree water incubation at room temperature for 30 minutes
15.09	Transformation of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]
	2 µL of ligation batch were transformed into E.coli DH5? according to heat shock protocol a lot of colonies grew on LB-Amp plates
16.09	Colony PCR of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]
	PCR mixture (50 µL total volume) 1 µL forward primer 1 µL reverse primer colony 2 µl MgCl2 1 µl dNTP Mix 1 µl DMSO 5 µl 10x Taq buffer 1 µl Taq-Polymerase 38 µl H2O template and primer specifications pSB1C3[LssmOrange]: lssmorange pprf + lssmorange ppr pSB1C3[mKate]: mkate pprf + mkate ppr PCR Program initial Denaturation 300s 95°C Denaturation 30s 95°C Annealing 30s 65°C Elongation 60s 72°C Final Elongation 300s 72°C analytical 1% agarose gel 2 positive clones for pPR-IBA2-LssmOrange 7 positive clones for pPR-IBA2-mKate
19.09	Plasmid prep
	using PureYield Plasmid Miniprep System (Promega) pPR-IBA2[LssmOrange] clone 5 c = 51,2 ng/µL pPR-IBA2[LssmOrange] clone 10 c = 55,5 ng/µL pPR-IBA2[mKate] clone 2 c = 32,3 ng/µL pPR-IBA2[mKate] clone 3 c = 35,2 ng/µL pPR-IBA2[mKate] clone 4 c = 29,1 ng/µL pPR-IBA2[mKate] clone 8 c = 37,6 ng/µL
19.09	Transformation in BL21DE3
	1 µL of plasmid was transformed into E.coli DH5? according to heat shock protocol biofilm grew on LB-Amp plates
21.09	Induction with IPTG
	pPR-IBA2[LssmOrange] and pPR-IBA2[mKate] clones were cultivated in SOC-Amp medium at 37°C and 150 rpm to an OD600 = 0,6 subsequent induction with IPTG (2 mM end concentration) and cultivation overnight at 30°C and 150 rpm induction worked for pPR-IBA2[LssmOrange] clone 10 and pPR-IBA2[mKate] clone 3 and clone 4
23.09	Spectral analysis of induced clones
	cells and cell lysate was analyzed using an fluorospectrometer
27.09	SDS-PAGE
	Gel of Biobrick producing cells M.Marker NEB Color Prestaind Broadrange 2.LssmOrange supernatant 3.mKate cells 4.Control (no visible band)