Team:Pasteur Paris/Week 12

Week 1 Week 2 Week 3 Week 4 Week 5 Week 6 Week 7 Week 8 Week 9 Week 10 Week 11 Week 12 Week 13 Week 14 Week 15

Week 12 08/17 - 08/21 NB-Esterase Assay MiniPrep 808030 in the plasmid pDG011. pNP-Assay Culture of DH5-α transformed with 808030 in pDG011 Enzymatic assay (pNP assay) of the first transformation (of the 11/08/15): BBa_808030 and pDG011 plasmid in DH5-α Gibson Assembly OD measurement of Cluster 4:   Concentration (ng/µL) OD(230nm) OD(260nm) OD(280nm) OD(260/230nm) OD(260/280nm) Cluster_4_start: 30 0.025 0.575 0.375 1.50 - /936011 55 0.4 1.075 0.575 1.88 2.65 936011/936023 7.5 0.6 0.125 0.05 2.41 0.21 936023/ 45 0.575 0.925 0.625 1.48 1.61 Cluster_4_end 47.5 0 0.925 0.575 1.64 - Gel migration of the PCR product of prom/pNB Est and Cluster_1_start. Gel plan: Stain 1 2 3 4 5 6 7 8 9 10 11 Tube number     A1 A2 A3 A4   B1 B2 B3 B4 Componants Ladder                     DNA concentration assay   Concentration (ng/µL) OD(230nm) OD(260nm) OD(280nm) OD(260/280nm) OD(260/230nm) Cluster 4 start 105 0.3 2.1 1.1 1.79 2.40 /936011 55 0 1.1 0.55 1.99 - 936011/936023 520 4.325 10.425 5.825 1.79 4.38 936023/ 47.5 0 0.925 0.5 1.89 - Cluster 4 end 130 0.6 2.625 1.475 1.79 4.38 Gibson Assembly Enzymatic digest of K936011, K936023 and K1392932 Enzymatic digest using Pst I and Spe I Gel Migration of digested plasmids K936011, K936023, K1392932. on a 0.8% agarose gel Gel Purification Cluster 4A Gibson assembly Gibson Assembly PCR amplification of cluster 4A Assembly. Gel migration on 0.8% agarose gel. Gel plan Well Number 1 2 3 4 5 Contains DNA ladder No primers Primer for only Primer rev only Both primers PCR amplification of Biobricks BBa_K808007, BBa_K808030, BBa_K936011, BBa_K936023, BBa_K1392932, BBa_K316003 PCR amplification using Phusion polymerase Gel Migration on a 1% agarose gel The PCR worked for most of our Biobricks with the exception of the biobrick K808030. DNA concentration measurement 1/25 dilution DNA concentration (ng/µl) OD(230nm) OD(260nm) OD(280nm) OD(340nm) OD(260/280nm) OD(260/230nm) 808007 4.3 0.08 0.085 0.052 0.006 1.64 1.06 808030 4.6 0.151 0.092 0.058 0.003 1.59 1.61 936011 5.9 0.141 0.118 0.073 0.009 1.61 0.83 936023 5.2 0.097 0.105 0.062 0.003 1.7 1.08 1392932 12.6 0.493 0.251 0.259 0.035 0.97 0.51 316003 5.5 0.1 0.11 0.062 0.0 1.76 1.10 PCR amplification of pSB1C3 PCR amplification using Phusion Polymerase. Gel Migration on a 1% agarose gel Well Number 1 2 3 4 5 6 7 Contains DNA ladder Non digested Plasmid Digested plasmid No primers Primer for only Primer rev only Both primers PCR amplification of pSB1C3 PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures.   No primers Primer for Primer rev   DNAse, RNAse free water 41.75 40.75 40.75 39.75 10X Ex Taq Buffer 5 5 5 5 dNTP 2 2 2 2 rescue_for 0 1 0 1 rescue_rev 0 0 1 1 DNA  Template 1 1 1 1 Ex Taq Polymerase 0.25 0.25 0.25 0.25 Total 50 50 50 50 Cycles: 95°C for 4min 25 cycles : 95°C for 40s annealing for 30s: 60°C: Tube 1-4 61.1°C: Tube 5-8 63°C: Tube 9-12 65.6°C: Tube 13-16 69.2°C: Tube 17-20 72.1°C: Tube 21-24 73.9°C: Tube 25-28 75°C: Tube 29-32 72°C for 80s 72°C for 7min. PCR amplification using Takara Ex Taq Gel Migration on a 1% agarose gel. PCR amplification of Biobricks K808030 PCR Amplification using Takara Ex Taq Polymerase. Gel migration on 1% Gel Plan: Well Number 1 2 3 4 5   DNA ladder No primers Primer For Primer Rev Extract PCR amplification of pSB1C3 PCR amplification using Takara Ex Taq Polymerase testing different annealing temperatures. Gel Migration on a 1% agarose gel. PCR amplification of pSB1C3 PCR amplification using Takara Ex Taq Polymerase testing 8 annealing temperatures.   No primers Primer for Primer rev   DNAse, RNAse free water 41.75 40.75 40.75 39.75 10X Ex Taq Buffer 5 5 5 5 dNTP 2 2 2 2 rescue_for 0 1 0 1 rescue_rev 0 0 1 1 DNA Template 1 1 1 1 Ex Taq Polymerase 0.25 0.25 0.25 0.25 Total 50 50 50 50 Gel Migration on a 1% Agarose Gel Enzymatic digest of BBa_K808030 Enzymatic digest by XbaI and Spe I of BBa_K808030 Gel migration. Yeast assembly The bacteria did not grow because the wrong antibiotic was chosen: BBa_J61047 was in pSB1A2 and not pSB1C3. Bacterial culture of BBa_J61047 on liquid LB + Amp 1X at 180 rpm, 37°C. The bacteria grew well. Midi-prep has been done with the 50 ml of BBa_J61047 culture. (dilution 1/1000)   DNA Concentration (ng/µl) OD(260nm) OD(280nm) OD(260/280nm) BBa_J61047 0.5 0.011 0.004 2.57 Preparation of YPD specific media on specific media (1% yeast extract, 2% dextrose, 2% peptone, 2% agar). Gel Migration of BBa_J61047. NotI enzymatic digest of BBa_J61047 in pSB1A2 enzyme. Gel Migration on a 0.8% agarose gel and the digestion hasn’t work. XbaI and SpeI digestion of BBa_J61047. tube 1 « Miniprep XbaI/SpeI » : 5 µl 10X buffer cutsmart + 0.5 µL XbaI + 0.5 µl SpeI + 38 µl H2O +5 µl DNA, digestion at 37°C during 1 hour. tube 2 « Midiprep XbaI/SpeI » : 5 µl 10X buffer cut smart + 0.5 µl XbaI + 0.5 µl SpeI + 38 µl H2O + 5 µl DNA, digestion at 37°C during 1 hour. tube 3 « Miniprep NotI » : 5 µl 10X buffer cutsmart + 0.5 µl NotI + 38.5 µL H2O + 5 µL DNA, digestion at 37°C during 1 hour. tube 4 « MiDiprep NotI » : 5 µl 10X buffer cutsmart + 0.5 µl NotI + 38.5 µL H2O + 5 µL DNA, digestion at 37°C during 1 hour. tube 5 « Miniprep CTRL » as negative control : 5 µl DNA + 40 µl H2O + 5 µl 10X buffer cutsmart. tube 6 « Midiprep CTRL » as negative control : 5 µl DNA + 40 µl H2O + 5 µl 10X buffer cutsmart. MWT : « Quick-Load Purple 2-Log DNA Ladder (0.1-10.0 kb) NEB PCR Amplification of the BBa_J61047 using Phusion Polymerase Master Mix. Gel Migration on a 0.8% agarose gel Culture of the plasmid pRS415 in SC_Leu at 30°C at 150 rpm for about 72 hours. PCR Amplification using the Phusion Polymerase of pRS415. Gel Extraction of the PCR products.  TPA solubility Determination of the solubilization products of TPA. Detection of 2-hydroxy-terephthalate using a TECAN spectrometer. Precipitation of terephthalic acid in H2SO4. ^ Page up