Team:Peking/Notebook/HPan
From 2010.igem.org
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===7.3=== | ===7.3=== | ||
Transformation of plasmid: Terminator(B0015) and T7 promoter(BBa_I719005) | Transformation of plasmid: Terminator(B0015) and T7 promoter(BBa_I719005) | ||
Line 211: | Line 211: | ||
Amplification of PbrR(PSB1K3) | Amplification of PbrR(PSB1K3) | ||
+ | |||
+ | ==August== | ||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.1|1]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.2|2]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.3|3]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.4|4]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.5|5]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.6|6]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.7|7]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.8|8]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#8.9|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.10|10]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.11|11]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.12|12]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.13|13]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.14|14]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.15|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.16|16]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.17|17]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.18|18]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.19|19]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.20|20]] | ||
+ | |style="text-align:center"| 21 | ||
+ | |- | ||
+ | |style="text-align:center"| 22 | ||
+ | |style="text-align:center"| 23 | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.24|24]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.25|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.26|26]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.27|27]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.28|28]] | ||
+ | |- | ||
+ | |style="text-align:center"| 29 | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.30|30]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#8.31|31]] | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | ===8.1=== | ||
+ | Miniprep:PbrR(PSB1K3) | ||
+ | |||
+ | Amplification of RBS+PbrR | ||
+ | ===8.2=== | ||
+ | Miniprep:RBS+PbrR | ||
+ | |||
+ | Digestion and identification by Electrophoresis | ||
+ | |||
+ | RBS+PbrR XbaI and PstI | ||
+ | |||
+ | Positive Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator again into strain BL21 | ||
+ | (DE3) | ||
+ | |||
+ | Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I) | ||
+ | ===8.3=== | ||
+ | Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | ===8.4=== | ||
+ | Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+phiR73 delta+Terminator+PO | ||
+ | promoter+GFP generator, but there is no colony of Pc promoter(1-18C)+RBS+PbrR | ||
+ | |||
+ | Digestion and identification by Electrophoresis, but the result had been failed. | ||
+ | |||
+ | N_B/B_X/B_SD/SD_B/PET21A/RBS+PbrR | ||
+ | |||
+ | Ligation of Pc promoter(1-18C) and RBS+PbrR again | ||
+ | |||
+ | Induce strain BL21(DE3)(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg | ||
+ | again to gain the expression of GFP, but there were still no obvious difference between strain induced and uninduced. | ||
+ | ===8.5=== | ||
+ | Bacterial colonies PCR | ||
+ | |||
+ | Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | |||
+ | Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | |||
+ | Transformation of Pc promoter(1-18C)+RBS+PbrR again. | ||
+ | |||
+ | Digestion and identification by Electrophoresis again | ||
+ | |||
+ | N_B/B_X/B_SD/SD_B/PET21A | ||
+ | |||
+ | Digestion and identification by Electrophoresis, but the result had been failed. | ||
+ | |||
+ | Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | |||
+ | Ligation of N_B, B_X(insert) and PET21A, B_SD, SD_B,PSB1K3 | ||
+ | |||
+ | Transformation of PET21A-NX and PSB3K3-BSD | ||
+ | |||
+ | Amplification of Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again | ||
+ | ===8.6=== | ||
+ | Amplification of Pc promoter(1-18C)+RBS+PbrR again | ||
+ | |||
+ | Bacterial colonies PCR again, but the result had been failed. | ||
+ | |||
+ | Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | |||
+ | Miniprep: Pc promoter(1-18C)+RBS+PbrR | ||
+ | |||
+ | Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again | ||
+ | |||
+ | Digestion and identification by Electrophoresis, but the result had been failed again. | ||
+ | |||
+ | Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | ===8.7=== | ||
+ | PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase for the second time | ||
+ | |||
+ | Digestion and identification by Electrophoresis, but the result had been failed again. | ||
+ | |||
+ | PbrR XbaI and PstI | ||
+ | |||
+ | RBS(B0034) SpeI and PstI | ||
+ | |||
+ | Ligation of PbrR(insert) and RBS(B0034)(vector) | ||
+ | ===8.8=== | ||
+ | Bacterial colonies PCR again, but the result had been failed. | ||
+ | |||
+ | PbrR+RBS | ||
+ | |||
+ | Amplification of RBS(B0034) | ||
+ | ===8.9=== | ||
+ | Digestion and identification by Electrophoresis | ||
+ | |||
+ | PbrR(PSB1K3) XbaI and PstI | ||
+ | |||
+ | RBS(B0034) SpeI and PstI | ||
+ | |||
+ | Ligation of PbrR(insert) and RBS(vector) for the second time | ||
+ | |||
+ | Transformation of RBS+PbrR again | ||
+ | |||
+ | ===8.10=== | ||
+ | Digestion and identification by Electrophoresis | ||
+ | |||
+ | PbrR(PCR product) XbaI and PstI | ||
+ | |||
+ | Ligation of PbrR(insert) and PSB1K3(vector) | ||
+ | |||
+ | Transformation fo PbrR(PSB1K3) | ||
+ | ===8.11=== | ||
+ | Bacterial colonies PCR , PbrR(PSB1K3) | ||
+ | |||
+ | PbrD/PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase | ||
+ | |||
+ | Indentification by Electrophoresis, but the PbrT had been failed. | ||
+ | |||
+ | PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase again. | ||
+ | |||
+ | Digestion and identification by Electrophoresis: PbrD and PSB1C3(backbone) | ||
+ | ===8.12=== | ||
+ | Digestion and identification by Electrophoresis: PbrR(PSB1K3)/PbrT | ||
+ | |||
+ | Positive transformation of RBS(B0034) | ||
+ | |||
+ | Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector) | ||
+ | |||
+ | Miniprep: RBS(B0034) | ||
+ | |||
+ | Digestion and identification by Electrophoresis: RBS(B0034) | ||
+ | |||
+ | ===8.13=== | ||
+ | Transformation of PbrD(PSB1C3), PbrT(PSB1C3), but there were no colony for them. | ||
+ | |||
+ | Ligation of PbrD/PbrT/PbrR(insert) and RBS(B0034)(vector) | ||
+ | ===8.14=== | ||
+ | PbrD/PbrT/PbrR PCR, 50 uL system with Easy Taq DNA Polymerase again, but the result of PbrT had been failed. | ||
+ | |||
+ | Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector) again | ||
+ | ===8.15=== | ||
+ | Amplification of PbrT+RBS and PbrD+RBS | ||
+ | |||
+ | Digestion and identification by Electrophoresis: PbrD and PbrR again | ||
+ | |||
+ | ===8.16=== | ||
+ | Miniprep: RBS+PbrT and RBS+PbrD | ||
+ | |||
+ | Digestion and identification by Electrophoresis: RBS+PbrT and RBS+PbrD | ||
+ | ===8.17=== | ||
+ | Miniprep: RBS+PbrT, RBS+PbrR and RBS+PbrD | ||
+ | |||
+ | Digestion and identification by Electrophoresis: RBS+PbrT, RBS+PbrR and RBS+PbrD | ||
+ | |||
+ | Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector) | ||
+ | |||
+ | Transformation of RBS+PbrT+Terminator and T7 promoter+RBS+PbrD | ||
+ | ===8.18=== | ||
+ | Digestion and identification by Electrophoresis:RBS+PbrR | ||
+ | ===8.19=== | ||
+ | Miniprep: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD | ||
+ | |||
+ | Digestion and identification by Electrophoresis: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD | ||
+ | ===8.20=== | ||
+ | Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) | ||
+ | ===8.24=== | ||
+ | Amplification of RBS+PbrR | ||
+ | |||
+ | Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) again | ||
+ | ===8.25=== | ||
+ | Miniprep: RBS+pbrR | ||
+ | |||
+ | Transformation of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator | ||
+ | |||
+ | Digestion and identification by Electrophoresis: RBS+PbrR | ||
+ | ===8.26=== | ||
+ | Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector) | ||
+ | ===8.27=== | ||
+ | Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | ===8.28=== | ||
+ | Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | |||
+ | Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator | ||
+ | ===8.30=== | ||
+ | Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 | ||
+ | promoter+RBS+Terminator+RBS+PbrT+Terminator | ||
+ | ===8.31=== | ||
+ | Ligation of MerP/PpbrA(insert) and CrtebI(vector) | ||
+ | |||
+ | ==September== | ||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.1|1]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.2|2]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.3|3]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.4|4]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.5|5]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.6|6]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.7|7]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.8|8]] | ||
+ | |style="text-align:center"| 9 | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.10|10]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.11|11]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.12|12]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.13|13]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.14|14]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.15|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.16|16]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|17]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|18]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|19]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|20]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.17-9.21|21]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.22|22]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.23|23]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.24|24]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.25|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.26|26]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.27|27]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#9.28|28]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#9.29|29]] | ||
+ | |style="text-align:center"| 30 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | ===9.1=== | ||
+ | Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into bacterium which consist of plasmid | ||
+ | PpbrA+RBS+GFP, but there is no expression of GFP | ||
+ | ===9.2=== | ||
+ | The sequencing results showed that the sequence of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 | ||
+ | promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect | ||
+ | |||
+ | PbrR PCR, 50 uL system with Easy Taq DNA Polymerase for the fourth time | ||
+ | |||
+ | Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third time | ||
+ | ===9.3=== | ||
+ | Retrieve the PCR product and identification by Electrophoresis | ||
+ | |||
+ | Transformation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third | ||
+ | |||
+ | Ligation of PbrR(insert) and RBS(vector) | ||
+ | ===9.4=== | ||
+ | Amplification of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator | ||
+ | |||
+ | Transformation of RBS+PbrR | ||
+ | ===9.5=== | ||
+ | Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator | ||
+ | |||
+ | Amplification of RBS+PbrR | ||
+ | |||
+ | Digestion and identification by Electrophoresis: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator | ||
+ | |||
+ | ===9.6=== | ||
+ | The sequencing results showed that the sequence of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect again | ||
+ | |||
+ | Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector) | ||
+ | |||
+ | Transformation of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD | ||
+ | ===9.7=== | ||
+ | Amplification of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD | ||
+ | ===9.8=== | ||
+ | Miniprep: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD | ||
+ | |||
+ | Digestion and identification by Electrophoresis: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD | ||
+ | ===9.10=== | ||
+ | Ligation of T7promoter+RBS+PbrD(insert) and RBS+PbrT+Terminator(vector). | ||
+ | |||
+ | Transformation of T7promoter+RBS+PbrD+RBS+PbrT+Terminator | ||
+ | ===9.11=== | ||
+ | Amplification of T7promoter+RBS+PbrD+RBS+PbrT+Terminator | ||
+ | |||
+ | Digestion and identification by Electrophoresis: RBS+PbrR | ||
+ | |||
+ | Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector) | ||
+ | ===9.12=== | ||
+ | Miniprep: T7promoter+RBS+PbrD+RBS+PbrT+Terminator | ||
+ | |||
+ | Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | |||
+ | Digestion and identification by Electrophoresis: T7promoter+RBS+PbrD+RBS+PbrT+Terminator | ||
+ | ===9.13=== | ||
+ | The sequencing result of T7promoter+RBS+PbrD+RBS+PbrT+Terminator had failed again. | ||
+ | |||
+ | Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | ===9.14=== | ||
+ | Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR | ||
+ | ===9.15=== | ||
+ | Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into PpbrA+GFP generator to detect the expression | ||
+ | level of GFP. But there were no expression of GFP. | ||
+ | ===9.16=== | ||
+ | The sequencing result showed than the sequence of PbrR was not intact. | ||
+ | ===9.17-9.21=== | ||
+ | Repeat the similar procedures to construct Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again. | ||
+ | ===9.22=== | ||
+ | Detect the expression level of GFP for the second time, but there were still no expression. | ||
+ | ===9.23=== | ||
+ | The sequencing result showed than the sequence of PbrR was not intact, which was similar to the sequence we had got before and Zhang Haoqian found that there was a PstI digest site in the sequence of PbrR | ||
+ | ===9.24=== | ||
+ | Transformation of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator into stain Trans 5a to detect the expression level of GFP | ||
+ | |||
+ | ===9.25=== | ||
+ | Ligation of Pc promoter+T7 polymerase(insert) and PSB3C5(backbone) | ||
+ | ===9.26=== | ||
+ | Miniprep: Pc promoter+T7 polymerase(PSB3C5) | ||
+ | |||
+ | Digestion and identification by Electrophoresis: Pc promoter+T7 polymerase(PSB3C5) | ||
+ | ===9.27=== | ||
+ | Transformation Pc promoter+T7 polymerase into bacterium with T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS | ||
+ | generator to detect the expression level of GFP | ||
+ | ===9.28=== | ||
+ | Amplification of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator and Pc promoter+T7 polymerase. | ||
+ | ===9.29=== | ||
+ | Detect the expression level of GFP | ||
+ | |||
+ | ==October== | ||
+ | |||
+ | {| class="calendar" border="0" rules="rows" width="650px" style="color:#ffffff" | ||
+ | |- | ||
+ | |style="text-align:center"| Mon | ||
+ | |style="text-align:center"| Tue | ||
+ | |style="text-align:center"| Wed | ||
+ | |style="text-align:center"| Thu | ||
+ | |style="text-align:center"| Fri | ||
+ | |style="text-align:center"| Sat | ||
+ | |style="text-align:center"| Sun | ||
+ | |- | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"|- | ||
+ | |style="text-align:center"|- | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.1|1]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.2|2]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.3|3]] | ||
+ | |- | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.4|4]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.5|5]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.6|6]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.7|7]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.8|8]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.9|9]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.10|10]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.11|11]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.12-10.13|12]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.12-10.13|13]] | ||
+ | |style="text-align:center"|[[Team:Peking/Notebook/HPan#10.14|14]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.15|15]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.16|16]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.17|17]] | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.18|18]] | ||
+ | |style="text-align:center"|19 | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.20|20]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.21|21]] | ||
+ | |style="text-align:center"|22 | ||
+ | |style="text-align:center"|23 | ||
+ | |style="text-align:center"| 24 | ||
+ | |- | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.25|25]] | ||
+ | |style="text-align:center"| [[Team:Peking/Notebook/HPan#10.26|26]] | ||
+ | |style="text-align:center"|27 | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |style="text-align:center"| - | ||
+ | |} | ||
+ | [<html><a href="#top">TOP</a></html>] | ||
+ | ===10.1=== | ||
+ | PCR T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS with the forword primer of T3 promoter and the univ reverse primer, 50 uL system. | ||
+ | |||
+ | Retrieve the PCR product of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS | ||
+ | ===10.2=== | ||
+ | Ligation of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(insert) and PSB1C3 bacbone | ||
+ | |||
+ | Transformation of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3) | ||
+ | ===10.3=== | ||
+ | Amplification of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3) | ||
+ | |||
+ | PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR with eight different Pc promoter | ||
+ | forward primer and PbrR reverse primer | ||
+ | |||
+ | Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR | ||
+ | |||
+ | ===10.4=== | ||
+ | Miniprep: T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3) | ||
+ | |||
+ | Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone) | ||
+ | |||
+ | Digestion and identification by Electrophoresis: T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3) | ||
+ | ===10.5=== | ||
+ | Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3 | ||
+ | (backbone), but there were no colonies for Pc promoter(1-18C)+RBS+PbrR | ||
+ | ===10.6=== | ||
+ | Bacterial colonies PCR and Amplification of Pc promoter(1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB1C3) | ||
+ | |||
+ | Ligation of RBS+PbrR(insert) and Pc promoter(1-18C)(vector) | ||
+ | |||
+ | Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB3K3(backbone) | ||
+ | ===10.7=== | ||
+ | Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB1C3) | ||
+ | |||
+ | Transformation of Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB3K3), Pc promoter(1-18C)+RBS+PbrR(PSB1C3) | ||
+ | ===10.8=== | ||
+ | Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB3K3), Pc promoter(1-18C)+RBS+PbrR(PSB1C3) | ||
+ | ===10.9=== | ||
+ | PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag with eight different Pc promoter | ||
+ | forward primer and Univ reverse primer | ||
+ | |||
+ | Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag, but the | ||
+ | product of Pc promoter(J23109) had been failed. | ||
+ | |||
+ | Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23112/J23114/J23117)+Pag(insert) and PSB1C3(backbone) | ||
+ | ===10.10=== | ||
+ | PCR Pc promoter(J23109)+Pag with Pc promoter(J23109) forward primer and Univ reverse primer | ||
+ | |||
+ | Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23112/J23114/J23117)+Pag( PSB1C3) | ||
+ | |||
+ | Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag( PSB1C3) | ||
+ | |||
+ | Retrieve the PCR product of Pc promoter(J231097)+Pag | ||
+ | ===10.11=== | ||
+ | Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag( PSB1C3) | ||
+ | |||
+ | Ligation of Pc promoter(J23109)+Pag(insert) and PSB1C3(backbone) | ||
+ | |||
+ | Transformation of Pc promoter(J23109)+Pag(PSB1C3) | ||
+ | ===10.12-13=== | ||
+ | Change the backbone of parts I had constructed from PSB1A3 to PSB1C3 | ||
+ | ===10.14=== | ||
+ | PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR with eight different Pc promoter forward primer and PbrR reverse primer for the second time | ||
+ | |||
+ | Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR | ||
+ | |||
+ | Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone) | ||
+ | |||
+ | Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone) | ||
+ | ===10.15=== | ||
+ | Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR( PSB1C3) | ||
+ | ===10.16=== | ||
+ | Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR( PSB1C3) | ||
+ | |||
+ | PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J 23114)+Pag with six different Pc promoter forward primer and Univ reverse primer | ||
+ | |||
+ | Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag | ||
+ | |||
+ | Ligation of Pc promoter Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag (insert)+PSB1C3(backbone) | ||
+ | |||
+ | Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3) | ||
+ | ===10.17=== | ||
+ | Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3), but | ||
+ | the amplification had faild. | ||
+ | ===10.18=== | ||
+ | Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3), but the amplification had faild again. | ||
+ | ===10.20=== | ||
+ | Ligation of Pc promoter Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag (insert)+PSB1C3(backbone) again | ||
+ | |||
+ | Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)For the second time | ||
+ | ===10.21=== | ||
+ | Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3) | ||
+ | ===10.25=== | ||
+ | Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3) | ||
+ | |||
+ | Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3) into strain with Psid-GFP to gain the expression of GFP | ||
+ | ===10.26=== | ||
+ | Detect the expression level of GFP in eight different strains | ||
+ | |||
+ | |||
+ | <html> | ||
+ | </div> | ||
+ | |||
+ | |||
+ | |||
+ | <div id="project description bottom"> | ||
+ | <div id="bottomgreen"> | ||
+ | <br> | ||
+ | <a href="https://2010.igem.org/Team:Peking/Team/HPan"><font color=#FFFFFF>==go to his page==</font></a> | ||
+ | </div> | ||
+ | </html> |
Latest revision as of 10:16, 27 October 2010
Contents
July
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
- | - | - | - | 1 | 2 | 3 |
4 | 5 | 6 | 7 | 8 | 9 | 10 |
11 | 12 | 13 | 14 | 15 | 16 | 17 |
18 | 19 | 20 | 21 | 22 | 23 | 24 |
25 | 26 | 27 | 28 | 29 | 30 | 31 |
[TOP]
7.3
Transformation of plasmid: Terminator(B0015) and T7 promoter(BBa_I719005)
7.4
PCR to standardize the MerT and MerP from plasmid NRI
Amplification of bacterium: Terminator(B0015) and T7 promoter(BBa_I719005)
7.5
Miniprep: Terminator(B0015) and T7 promoter(BBa_I719005)
Digestion and identification by Electrophoresis
Terminator(B0015) EcoRI and XbaII
T7 promoter(BBa_I719005) SpeI and PstI
phiR73 delta(BBa_I746352) EcoRI and SpeI
PO promoter(BBa_I746361) EcoRI and XbaI
7.6
Ligation of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
Transformation of ligation mixture of phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
7.7
Amplification of bacterium: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
Miniprep: phiR73 delta(BBa_I746352)(insert) and Terminator(B0015)(vector)
7.8
Digestion and identification by Electrophoresis, but the result had been failed.
phiR73 delta+Terminator XbaI and PstI
RBS(B0032) SpeI and PstI
Digestion and identification by Electrophoresis again
7.9
Ligation of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)
Transformation of ligation mixture of phiR73 delta+Terminator(insert) and RBS(B0032)(vector)
Digestion and identification by Electrophoresis
Terminator(B0015) XbaI and PstI
AraC XbaI and PstI
Miniprep: plasmid of backbone PSB1C3
7.10
Amplification of RBS+phiR73 delta+Terminator
7.11
The amplification had failed and amplification again.
7.12
Miniprep: phiR73 RBS+delta+Terminator
Digestion and identification by Electrophoresis
RBS+phiR73 delta+Terminator XbaI and PstI
7.13
Ligation of RBS+phiR73 delta+terminator(insert) and T7 promoter((BBa_I719005))(vector)
Transformation of ligation mixture of RBS+phiR73 delta+Terminator and T7 promoter((BBa_I719005))(vector)
7.14
Amplification of T7 promoter+RBS+phiR73 delta+Terminator
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator
Digestion and identification by Electrophoresis
T7 promoter+RBS+phiR73 delta+Terminator EcoRI and SpeI
7.15
Ligation of T7 promoter+RBS+phiR73 delta+Terminator(insert) and PO promoter(BBa_I746361)(vector)
Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
7.16
Amplification of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter
7.17
Digestion and identification by Electrophoresis
T7 promoter+RBS+phiR73 delta+Terminator+PO promoter EcoRI and SpeI
7.25
Digestion and identification by Electrophoresis
GFP generator(E0840) EcoRI and Xbal
Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter(insert) and GFP generato T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator r(E0840)(vector)
Transformation of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
7.26
Amplification of Ligation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
7.27
Miniprep: T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
Digestion and identification by Electrophoresis
T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator
7.28
Positive transformation T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator into strain BL21a
7.29
PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase
Induce strain BL21a(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg to gain the expression of GFP, but there were no obvious difference between strain induced and uninduced.
7.30
Retrieve the PCR product and identification by Electrophoresis
Digestion:
PbrR EcoRI and PstI/XbaI and PstI
Ligation of PbrR(insert) and PSB1K3(vector)
Transformation of PbrR(PSB1K3)
7.31
Ligation of PbrR(insert) and RBS(B0034)
Transformation of RBS+PbrR
Amplification of PbrR(PSB1K3)
August
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
1 | 2 | 3 | 4 | 5 | 6 | 7 |
8 | 9 | 10 | 11 | 12 | 13 | 14 |
15 | 16 | 17 | 18 | 19 | 20 | 21 |
22 | 23 | 24 | 25 | 26 | 27 | 28 |
29 | 30 | 31 | - | - | - | - |
[TOP]
8.1
Miniprep:PbrR(PSB1K3)
Amplification of RBS+PbrR
8.2
Miniprep:RBS+PbrR
Digestion and identification by Electrophoresis
RBS+PbrR XbaI and PstI
Positive Transformation of T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator again into strain BL21 (DE3)
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)
8.3
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
8.4
Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator, but there is no colony of Pc promoter(1-18C)+RBS+PbrR
Digestion and identification by Electrophoresis, but the result had been failed.
N_B/B_X/B_SD/SD_B/PET21A/RBS+PbrR
Ligation of Pc promoter(1-18C) and RBS+PbrR again
Induce strain BL21(DE3)(consist of plasmid T7 promoter+RBS+phiR73 delta+Terminator+PO promoter+GFP generator) by iptg again to gain the expression of GFP, but there were still no obvious difference between strain induced and uninduced.
8.5
Bacterial colonies PCR
Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
Transformation of Pc promoter(1-18C)+RBS+PbrR again.
Digestion and identification by Electrophoresis again
N_B/B_X/B_SD/SD_B/PET21A
Digestion and identification by Electrophoresis, but the result had been failed.
Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
Ligation of N_B, B_X(insert) and PET21A, B_SD, SD_B,PSB1K3
Transformation of PET21A-NX and PSB3K3-BSD
Amplification of Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again
8.6
Amplification of Pc promoter(1-18C)+RBS+PbrR again
Bacterial colonies PCR again, but the result had been failed.
Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
Miniprep: Pc promoter(1-18C)+RBS+PbrR
Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again
Digestion and identification by Electrophoresis, but the result had been failed again.
Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
8.7
PbrR PCR, 50 uL system with Easy Pfu DNA Polymerase for the second time
Digestion and identification by Electrophoresis, but the result had been failed again.
PbrR XbaI and PstI
RBS(B0034) SpeI and PstI
Ligation of PbrR(insert) and RBS(B0034)(vector)
8.8
Bacterial colonies PCR again, but the result had been failed.
PbrR+RBS
Amplification of RBS(B0034)
8.9
Digestion and identification by Electrophoresis
PbrR(PSB1K3) XbaI and PstI
RBS(B0034) SpeI and PstI
Ligation of PbrR(insert) and RBS(vector) for the second time
Transformation of RBS+PbrR again
8.10
Digestion and identification by Electrophoresis
PbrR(PCR product) XbaI and PstI
Ligation of PbrR(insert) and PSB1K3(vector)
Transformation fo PbrR(PSB1K3)
8.11
Bacterial colonies PCR , PbrR(PSB1K3)
PbrD/PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase
Indentification by Electrophoresis, but the PbrT had been failed.
PbrT PCR, 50 uL system with Easy Pfu DNA Polymerase again.
Digestion and identification by Electrophoresis: PbrD and PSB1C3(backbone)
8.12
Digestion and identification by Electrophoresis: PbrR(PSB1K3)/PbrT
Positive transformation of RBS(B0034)
Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector)
Miniprep: RBS(B0034)
Digestion and identification by Electrophoresis: RBS(B0034)
8.13
Transformation of PbrD(PSB1C3), PbrT(PSB1C3), but there were no colony for them.
Ligation of PbrD/PbrT/PbrR(insert) and RBS(B0034)(vector)
8.14
PbrD/PbrT/PbrR PCR, 50 uL system with Easy Taq DNA Polymerase again, but the result of PbrT had been failed.
Ligation of PbrD(insert) and PSB1C3(vector), PbrT(insert) and PSB1C3(vector) again
8.15
Amplification of PbrT+RBS and PbrD+RBS
Digestion and identification by Electrophoresis: PbrD and PbrR again
8.16
Miniprep: RBS+PbrT and RBS+PbrD
Digestion and identification by Electrophoresis: RBS+PbrT and RBS+PbrD
8.17
Miniprep: RBS+PbrT, RBS+PbrR and RBS+PbrD
Digestion and identification by Electrophoresis: RBS+PbrT, RBS+PbrR and RBS+PbrD
Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector)
Transformation of RBS+PbrT+Terminator and T7 promoter+RBS+PbrD
8.18
Digestion and identification by Electrophoresis:RBS+PbrR
8.19
Miniprep: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD
Digestion and identification by Electrophoresis: RBS+PbrT+Terminator and T7 promoter+RBS+PbrD
8.20
Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector)
8.24
Amplification of RBS+PbrR
Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) again
8.25
Miniprep: RBS+pbrR
Transformation of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
Digestion and identification by Electrophoresis: RBS+PbrR
8.26
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector)
8.27
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
8.28
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
8.30
Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
8.31
Ligation of MerP/PpbrA(insert) and CrtebI(vector)
September
Mon | Tue | Wed | Thu | Fri | Sat | Sun |
- | - | 1 | 2 | 3 | 4 | 5 |
6 | 7 | 8 | 9 | 10 | 11 | 12 |
13 | 14 | 15 | 16 | 17 | 18 | 19 |
20 | 21 | 22 | 23 | 24 | 25 | 26 |
27 | 28 | 29 | 30 | - | - | - |
[TOP]
9.1
Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into bacterium which consist of plasmid PpbrA+RBS+GFP, but there is no expression of GFP
9.2
The sequencing results showed that the sequence of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR and T7 promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect
PbrR PCR, 50 uL system with Easy Taq DNA Polymerase for the fourth time
Liagation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third time
9.3
Retrieve the PCR product and identification by Electrophoresis
Transformation of T7 promoter+RBS+Terminator(insert)+RBS+PbrT+Terminator(vector) for the third
Ligation of PbrR(insert) and RBS(vector)
9.4
Amplification of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
Transformation of RBS+PbrR
9.5
Miniprep: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
Amplification of RBS+PbrR
Digestion and identification by Electrophoresis: T7 promoter+RBS+Terminator+RBS+PbrT+Terminator
9.6
The sequencing results showed that the sequence of T7 promoter+RBS+Terminator+RBS+PbrT+Terminator was incorrect again
Ligation of RBS+PbrT(insert) and Terminator(B0015), RBS+PbrD(insert) and T7 promoter(vector)
Transformation of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
9.7
Amplification of RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
9.8
Miniprep: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
Digestion and identification by Electrophoresis: RBS+PbrT+Terminator, T7 promoter+RBS+PbrD
9.10
Ligation of T7promoter+RBS+PbrD(insert) and RBS+PbrT+Terminator(vector).
Transformation of T7promoter+RBS+PbrD+RBS+PbrT+Terminator
9.11
Amplification of T7promoter+RBS+PbrD+RBS+PbrT+Terminator
Digestion and identification by Electrophoresis: RBS+PbrR
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)(vector)
9.12
Miniprep: T7promoter+RBS+PbrD+RBS+PbrT+Terminator
Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
Digestion and identification by Electrophoresis: T7promoter+RBS+PbrD+RBS+PbrT+Terminator
9.13
The sequencing result of T7promoter+RBS+PbrD+RBS+PbrT+Terminator had failed again.
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
9.14
Digestion and identification by Electrophoresis: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR
9.15
Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR into PpbrA+GFP generator to detect the expression level of GFP. But there were no expression of GFP.
9.16
The sequencing result showed than the sequence of PbrR was not intact.
9.17-9.21
Repeat the similar procedures to construct Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I)+RBS+PbrR again.
9.22
Detect the expression level of GFP for the second time, but there were still no expression.
9.23
The sequencing result showed than the sequence of PbrR was not intact, which was similar to the sequence we had got before and Zhang Haoqian found that there was a PstI digest site in the sequence of PbrR
9.24
Transformation of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator into stain Trans 5a to detect the expression level of GFP
9.25
Ligation of Pc promoter+T7 polymerase(insert) and PSB3C5(backbone)
9.26
Miniprep: Pc promoter+T7 polymerase(PSB3C5)
Digestion and identification by Electrophoresis: Pc promoter+T7 polymerase(PSB3C5)
9.27
Transformation Pc promoter+T7 polymerase into bacterium with T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator to detect the expression level of GFP
9.28
Amplification of T7 promoter+RBS+phiR73 delat+Terminator+PO promoter+RBS generator and Pc promoter+T7 polymerase.
9.29
Detect the expression level of GFP
October
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[TOP]
10.1
PCR T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS with the forword primer of T3 promoter and the univ reverse primer, 50 uL system.
Retrieve the PCR product of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS
10.2
Ligation of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(insert) and PSB1C3 bacbone
Transformation of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
10.3
Amplification of T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR with eight different Pc promoter forward primer and PbrR reverse primer
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR
10.4
Miniprep: T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone)
Digestion and identification by Electrophoresis: T3 promoter+RBS+phiR73 delta+Terminator+PO promoter+RBS(PSB1C3)
10.5
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3 (backbone), but there were no colonies for Pc promoter(1-18C)+RBS+PbrR
10.6
Bacterial colonies PCR and Amplification of Pc promoter(1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB1C3)
Ligation of RBS+PbrR(insert) and Pc promoter(1-18C)(vector)
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB3K3(backbone)
10.7
Miniprep: Pc promoter(1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB1C3)
Transformation of Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB3K3), Pc promoter(1-18C)+RBS+PbrR(PSB1C3)
10.8
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(PSB3K3), Pc promoter(1-18C)+RBS+PbrR(PSB1C3)
10.9
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag with eight different Pc promoter forward primer and Univ reverse primer
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag, but the product of Pc promoter(J23109) had been failed.
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23112/J23114/J23117)+Pag(insert) and PSB1C3(backbone)
10.10
PCR Pc promoter(J23109)+Pag with Pc promoter(J23109) forward primer and Univ reverse primer
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23112/J23114/J23117)+Pag( PSB1C3)
Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag( PSB1C3)
Retrieve the PCR product of Pc promoter(J231097)+Pag
10.11
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+Pag( PSB1C3)
Ligation of Pc promoter(J23109)+Pag(insert) and PSB1C3(backbone)
Transformation of Pc promoter(J23109)+Pag(PSB1C3)
10.12-13
Change the backbone of parts I had constructed from PSB1A3 to PSB1C3
10.14
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR with eight different Pc promoter forward primer and PbrR reverse primer for the second time
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR
Ligation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone)
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR(insert) and PSB1C3(backbone)
10.15
Bacterial colonies PCR and Amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR( PSB1C3)
10.16
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J23112/J23114/J23117)+RBS+PbrR( PSB1C3)
PCR Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/J 23114)+Pag with six different Pc promoter forward primer and Univ reverse primer
Retrieve the PCR product of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag
Ligation of Pc promoter Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag (insert)+PSB1C3(backbone)
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)
10.17
Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3), but the amplification had faild.
10.18
Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3), but the amplification had faild again.
10.20
Ligation of Pc promoter Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag (insert)+PSB1C3(backbone) again
Transformation of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)For the second time
10.21
Bacterial colonies PCR and amplification of Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)
10.25
Miniprep: Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3)
Transformation Pc promoter(1-18C/1-18E/2-2E/1-20M/1-18I/J23109/ J23114)+Pag(PSB1C3) into strain with Psid-GFP to gain the expression of GFP
10.26
Detect the expression level of GFP in eight different strains