Team:Westminster UK/HP

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Lab note book for IGEM 15/09/17 Week off labs 23/09/17-24/09/17 -Made 3L LB broth -Autoclaved tips and Eppendorf tubes - Inoculation of pSB1C3 with YFP +RFP First characterisation of pSB1C3 with RFP Number Content Strain 1 mRFPG no IPTG TOP 10 2 mRFPG + IPTG TOP 10 3 mRFPB no IPTG DH5α 4 mRFPB + IPTG DH5α 5 YFP no IPTG TOP 10 6 YFP + IPTG TOP 10 7 mRFPG + mRFPB no IPTG TOP 10 + DH5α 8 mRFPG + mRFPB + IPTG TOP 10 + DH5α 25/09/17 -Made 400ml LB broth - Miniprep of pSB1C3 containing YFP and MRFP Content Concentration (ng/µl) 260/280 ratio YFP 1 169.2 1.76 YFP 2 166.6 1.85 MRFP 3 179.0 1.85 MRFP 4 190.0 1.72 Digestion of YFP samples: - Eppendorf 1 (well 1): MRFP 1 cut with Ecor1 and Pst1 - Eppendorf 2 (well 2): MRFP 2 cut with Xba1 and Pst1 - Eppendorf 3 (well 3): MRFP 1 cut with Ecor1 and Pst1 - Eppendorf 4 (well 4): MRFP 2 cut with Xba1 and Pst1 Samples run on a 1% agarose gel Samples extracted from gel and stored at -20°C 27/09/17 Characterising part Number Content Strain Absorption 1 mRFPG no IPTG DH5α 1.568 2 mRFPG + IPTG DH5α 1.556 3 mRFPB no IPTG TOP 10 1.379 4 mRFPB + IPTG TOP 10 1.617 5 YFP no IPTG TOP 10 0.081 6 YFP + IPTG TOP 10 0.084 7 mRFPG + mRFPB no IPTG TOP 10 + DH5α 1 8 mRFPG + mRFPB + IPTG TOP 10 + DH5α 1.474 9 YFP no antibiotic no IPTG TOP 10 1.175 10 MRFPG no antibiotic +IPTG TOP 10 1.525 11 Control Nothing 1 Quantifying gel extracts: Content Concentration (ng/µl) 260/280 ratio YFP 1 vec X 19.8 2.01 YFP 1 vec E 11.7 1.69 YFP1 frag x 3.9 2.56 YFP1 frag E 6.6 1.93 YFP vec 2 X 2 2.0 3.26 YFP 2 vec E 1 6.9 2.15 YFP2 vec E 2 2.3 6.33 YFP 2 frag X 1 3.3 3.47 YFP 2 frag X 2 2.4 2.90 YFP 2 frag E 1 10.9 3.32 YFP2 E frag 2 6.2 2.40 Quantifying Mrfp: Content Concentration (ng/µl) 260/280 ratio Mrfp 3 vec 2 2.08 73.0 Mrfp 3 vec 3 3.7 8.12 Rsal 1 18.6 2.16 Rsal 2 10.7 2.13 MRFP 4 frag 2 137.9 1.68 YFP frag 1 8.6 3.50 YFP frag 2 11.8 3.14 YFP vec 1 6.7 2.36 YFp vec 2 4.9 7.57 Mrfp 3 frag 19.6 6.46 Mrfp vec 2 1 9.5 2.17 Mrfp vec 2 2 13.7 2.13 YFP 3 vec 1 19.3 2.19 Digestion: -Eppendorf 1: MRFP3 + Ecor1 + Pst1 -Eppendorf 2: MRFP 4 + Ecor 1 + Pst1 Ligation: Ligated on XL-1b blue plates 29/09/17 Miniprep of Rsal colonies Content Concentration (ng/µl) 260/280 ratio Rsal 1 216.7 1.88 Rsal 2 287.5 1.82 2/10/17 - Sub-culturing Rsal colonies on a new plat - Made 500 ml LB broth and plated 04//10/17 Digestion: Eppendorf 1: Rsal 1 construct cut with Ecor1 + PST1 Eppendorf 2: Rsal 2 construct cut with NOT1 05/10/17 -Re-suspended LapG from IDT to create a 10 ng/µl Digestion: Eppendorf 1: ppuR with Xba1 and PST1 Eppendorf 2: LapG with Ecor1 and Spe1 Eppendorf 3: Psoa with Ecor1 and Spe1 Ligation: Eppendorf 1: ppuR and YFP vector X Eppendorf 2: Psoa + YFP2 vec E1 +MRFP 4 frag 2 Eppendorf 3: LapG + YFP2 vec E1 +MRFP 4 frag 2 Transformation on TOP 10, SCS and XL-1 blue competent cells, no growth seen. 11/10/17 Miniprep of Psoa Content Concentration (ng/µl) 260/280 ratio Psoa 1 366.6 1.89 Psoa 2 149.8 1.89 Digestion: Eppendorf 1: Psoa 2 with Ecor1 and not 1 Eppendorf 2: Psoa 1 with Ecor 1 and Xba1 1% gel ran at 80 volts for 80 minutes. Ligation: Psoa1 + pSB1C3 containing ppuR Transformation using TOP 10 competent cells 12/10/17 Inoculation of colonies containing Rsal, ppuI and ppuR 13/10/17 Miniprep of colonies containing ppuI, ppuR and Rsal Results: Content Concentration (ng/µl) 260/280 ratio ppuI 1 55.2 1.95 ppuI2 204.8 1.82 ppuR 1 53.8 1.92 ppuR 2 88.8 1.90 Rsal 1 103.4 1.95 Rsal 2 152.8 1.88 14/10/17 Digestion: 1) LapG with Ecor1, Xba1 2) psoa promoter with Xba1 and Spe1 3) Rsal with NOT1 4 ppuI with NOT1 5) ppuR with Xba1 and Spe1 6) ppuR with Ecor1 and Xba1 Ligation: Eppendorf 1: LapG with appropriate cut vector Eppendorf 2: ppuR (Ecor1 and Xba1) with Psoa promoter Transformation: 1ul +3ul DNA into 50ul and 100ul TOP 10 competent cells 1% gel carried out 15/10/17 • Gel purification was performed on extracted pSB1C3 vector. Vector fragments weighed 0.154 g and 0.122 g respectively • Rsal gene digested with NOT1 while ppuR was digested with Ecor1 and Xba1 • Ligation using extracted pSB1C3 vector and genes • Transformation using TOP10 competent cells. • Inoculation of LapG, ppuR into 10ml of LB media Gel did not work. 17/10/17 • Transformation of 50ul Rosetta-Gami competent cells with pSB1C3 paired with BBa_J04450. 18/10/17 • Inoculation of Rosetta-Gami bacterial colonies • Characterisation of TOP 10 rfp expression 19/10/17 • Miniprep performed on inoculated colonies Content Concentration (ng/µl) 260/280 ratio Rosseta 1 75 1.83 Rosseta 2 84 2.06 • Digestion with EcoR1 +Pst1, digestion with EcoR1 20/10/17 • 1 % gel ran • Rosetta EcoR1 + Xba1 fragment extracted. 21/10/17 • Lap G digested with EcoR1 + Xba1 • Ligated with extracted Rosetta pSB1C3 • Transformation of Lap G construct with 50ul Xl-1 blue competent cells 22/10/17 • Inoculation of ppuI, RsaL, LapG 23/10/17 • Miniprep of inoculants: Content Concentration (ng/µl) 260/280 ratio ppuI 1 159 1.87 ppuI2 194 1.81 Rsal 1 215 1.92 Rsal 2 183 1.95 Lap G 1 120 1.93 Lap G 2 233 1.77 DNA was diluted with dh20 before being packed off. 24/10/17 • Digestion of Lap G with Ecor1 +Xba1 • 1% agarose gel ran •