Toggle navigation SCU-Igem Project Background Overview Description Result Modeling Human Practice Presentation at Seventh Senior High School Visiting University of Electronic Science and Technology of China (UESTC) Safety Parts Attributions Team Notebook Notebook Notebook of Biobricks Notebook of Transmitter Notebook of Effector Notebook of Sensor Method Bacterial Genomic DNA Prep Digestion Gel Extraction Linkage PCR Plasmid Mini Prep The Notebook of Sensor Back to top Week 1 8.21-8.24 Week 2 8.25-8.31 Since our team are sorted into 4 groups our target is constructing the Sensor cell in our system. According to our design, we need to construct two gene pathways. Consequently, we use the following parts: pLac (BBa_R0080), Promoter C and arabinose C operon (BBa_I13458), RBS (BBa_B0030), LuxI (BBa_C0061), double terminator (BBa_B0015), Constitutive promoter (BBa_J23100), CinI (BBa_C0076), pBAD (BBa_K206000), and LacI (BBa_C0012).Week 1 7.12-7.191. We transformed the BBa_B0015, BBa_C0061, and BBa_B0030 plasmids into E.coli.2. We extracted the plasmids and tested them by electrophoresis.ResultsThe concentration of plasmidsName Concentration (ng/3μl) BBa_B0015 118/59.5 BBa_B0030 23.7/23.2 BBa_C0061 260/161.9 Note: The different concentrations are from different tubes. Week 2 7.20-7.26- 1. We transformed the BBa_K084012, BBa_I761014, BBa_K081005, BBa_J23100, BBa_K206000, BBa_R0080, BBa_C0076, BBa_C1002, and BBa_I13458 which are tested by cleavage and electrophoresis.ResultsThe concentration of plasmidsName Concentration (ng/3μl) BBa_K084012 26/35.4 BBa_I761014 49/68 BBa_K081005 32.2/22 BBa_J23100 120/75.7 BBa_K206000 58.7 BBa_R0080 Failed BBa_C0076 98.6 BBa_C1002 85.7 BBa_I13458 496.6/242.6 Note: The different concentrations are from different tubes.The electrophoresis results are shown in Figure 1. Figure 1. The plasmids that has been cleaved by enzyme. Week 3 7.27-8.031. We cleaved the BBa_K084012, BBa_C0012, BBa_I13458 with EcoR I and Spe I, BBa_I761014, and BBa_K206000 with Xba I and Pst I.2. We amplified the BBa_K084012 and BBa_K081005 by transforming them into E.coli. And then we extracted the plasmids and tested them by electrophoresis.3. We linked the BBa_K084012 and BBa_B0015; BBa_I13458 and BBa_K206000; BBa_K081005 and BBa_C1002 with pSB1A3.ResultsThe concentration of plasmidsName Concentration (ng/3μl) BBa_K084012 95.74/37.18 BBa_K081005 51.24/27.16 Note: The different concentrations are from different tubes.2. The electrophoresis results are shown in Figure 2. Figure 2. The plasmids that have been cleaved by EcoR I and Spe I or Xba I and Pst I.Week 4 8.04-8.101. We re-cleaved the BBa_K081005 for linkage (BBa_K081005 and BBa_C0012 with pSB1A3).2. We transformed the linkage products into E.coli. Then we extracted the plasmids to test their qualities.3. We transformed, extracted, and tested (BBa_K081005 and BBa_C0012 with pSB1A3).4. We sent some of our linkage products to sequence them.5. We extracted the BBa_B0015 and BBa_J04656. We cleaved the BBa_B0015 (E,X), BBa_K084012 PLUS BBa_B0015 (E, S), BBa_K084012 (E, S), BBa_K081005 (S,P), and BBa_C1002 (X,P) and we did the gel purification.7. We amplified the BBa_I761014 by PCR.ResultsThe concentration of plasmidsName Concentration BBa_J0465 123.6/123.2 BBa_B0015 40/48/34 Note: The different concentrations are from different tubes.2. The electrophoresis results are shown in Figure 3. Figure 3. A and C show the plasmids that have been cleaved. B shows the PCR products.Week 5 8.11-8.171. We linked the BBa_K081005 and BBa_C1002; BBa_I13458 PLUS BBa_K206000 and PCR products (Traditional Assembly); BBa_K084012 and BBa_B0015; BBa_K081005 and BBa_C1002 (3A Assembly) and transformed them.2. We extracted the plasmids and did the linkage again (3A assembly).3. We cleaved the BBa_K084012 (E, S) (Named Cut 1), BBa_B0015 (X, P) (Cut2), BBa_I13458 PLUS BBa_K206000 (E, S) (Cut3), PCR products (X, P) (Cut4), BBa_K81005 (E, S) (Cut5), BBa_C1002 (X, P) (Cut6).We linked the Cut1 and Cut2 (Named Lia1); Cut3 and Cut4 (Lia 2 and 3); Cut5 and Cut6 (Lia 4). Results1. The electrophoresis results are shown in Figure 4. Figure 4. The parts that have been cleaved for linkageWeek 6 8.18-8.241. We cleaved the BBa_K081005 (E, S) and BBa_C1002 (X, P) plasmids.2. We tested the lia 6-1-1 (X, P), lia 7-1-1 (X, P), lia 7-1-2 (X, P), lia 8-2-1 (E, S), and lia 8-2-2 (E, S) by cleavage and electrophoresis.3. We did the gel purification for all the cleavage products.4. We cleaved the BBa_K081005 (E, S), lia 6-1-4 (X, P), lia 6-1-1 (X, P), lia 6-1-2 (X, P), lia 6 -1-5 (X, P), lia 4-2-2 (E, S) and control group (E, S). Also, we prepared the linear backbones (pSB1T3 and pSB1C3) by cleavage.5. We linked the CUT7 and CUT8 with pSB1T3 (Named NR 1), CUT3 and CUT4 with pSB1C3, pSB1T3, and pSB1K3, BBa_K081005 and CUT6 with pSB1T3 (NR 2).6. We transformed the linkage products and extracted the BBa_I761014 and BBa_K084012 plasmids.7. We cleaved the BBa_I761014 and BBa_C1002 with Xba I and Pst I. And we tested them by electrophoresis.8. We amplified BBa_I761014 by PCR.ResultsThe concentration of plasmidsName Concentration (ng/3 μl) BBa_I761014 40 BBa_C0012 24 2. The electrophoresis results are shown in Figure 5. Figure 5. A. gel purification result; B and C. the plasmids that have been cleaved by enzyme. D. PCR results. Week 7 8.25-8.311. We tested the quality of our PCR products by electrophoresis.2. We extracted the NR1-1, NR1-2, NR1-3, NR1-4, NR1-5, NR1-6, NR2-1, NR2-2, NR2-3, NR2-4, NR2-5, and NR2-6.3. We tested BBa_B0015 (X, P), BBa_I13458 PLUS BBa_K206000 (E, S), BBa_C0012 (S, P), BBa_K084012 (X, P), NR1-1 (X, P), NR1-2 (X, P), NR2-1 (E, S), NR2-2 (E, S) by cleavage.4. We cleaved NR2-3, NR2-5, NR2-6 with EcoR I and Spe I.5. We linked the BBa_I13458 PLUS BBa_K20600 and our PCR products with pSB1K3 and pSB1C3.6. We transformed the linkage products.7. We tested the NR1-3, NR1-4 (E, S) and NR2-1/NR2-6 (X, P).8. We amplified BBa_I761014 by PCR and did the PCR purification9. We extracted NR3 and BBa_I761014 and tested them by electrophoresis and cleavage.10. We linked BBa_I13458 PLUS BBa_K206000 and NP2 with pSB1T3 and pSB1C3.11. We linked BBa_I13458 PLUS BBa_K206000 and BBa_I761014 with pSB1T3, BBa_0030 and BBa_C0076 with pSB1A3 and pSB1T3.ResultsThe concentration of plasmidsName Concentration (ng/3 μl) NR1-1 95.5 NR1-2 127 NR1-3 105 NR1-4 128 NR1-5 128 NR1-6 110 NR2-1 112 NR2-2 138 NR2-3 135 NR2-4 128 NR2-5 102 NR2-6 Failed The electrophoresis results are shown in Figure 6. Figure 6. A. the results of cleaved plasmids and PCR products. B. and C. the results of linkage products. Sichuan university
The Notebook of
Sensor
Since our team are sorted into 4 groups our target is constructing the Sensor cell in our system. According to our design, we need to construct two gene pathways. Consequently, we use the following parts: pLac (BBa_R0080), Promoter C and arabinose C operon (BBa_I13458), RBS (BBa_B0030), LuxI (BBa_C0061), double terminator (BBa_B0015), Constitutive promoter (BBa_J23100), CinI (BBa_C0076), pBAD (BBa_K206000), and LacI (BBa_C0012).
Week 1 7.12-7.19
1. We transformed the BBa_B0015, BBa_C0061, and BBa_B0030 plasmids into E.coli.
2. We extracted the plasmids and tested them by electrophoresis.
Results
Name
Concentration (ng/3μl)
BBa_B0015
118/59.5
BBa_B0030
23.7/23.2
BBa_C0061
260/161.9
Note: The different concentrations are from different tubes.
Week 2 7.20-7.26-
1. We transformed the BBa_K084012, BBa_I761014, BBa_K081005, BBa_J23100, BBa_K206000, BBa_R0080, BBa_C0076, BBa_C1002, and BBa_I13458 which are tested by cleavage and electrophoresis.
BBa_K084012
26/35.4
BBa_I761014
49/68
BBa_K081005
32.2/22
BBa_J23100
120/75.7
BBa_K206000
58.7
BBa_R0080
Failed
BBa_C0076
98.6
BBa_C1002
85.7
BBa_I13458
496.6/242.6
The electrophoresis results are shown in Figure 1.
Figure 1. The plasmids that has been cleaved by enzyme.
Week 3 7.27-8.03
1. We cleaved the BBa_K084012, BBa_C0012, BBa_I13458 with EcoR I and Spe I, BBa_I761014, and BBa_K206000 with Xba I and Pst I.
2. We amplified the BBa_K084012 and BBa_K081005 by transforming them into E.coli. And then we extracted the plasmids and tested them by electrophoresis.
3. We linked the BBa_K084012 and BBa_B0015; BBa_I13458 and BBa_K206000; BBa_K081005 and BBa_C1002 with pSB1A3.
95.74/37.18
51.24/27.16
2.
Figure 2. The plasmids that have been cleaved by EcoR I and Spe I or Xba I and Pst I.
Week 4 8.04-8.10
1. We re-cleaved the BBa_K081005 for linkage (BBa_K081005 and BBa_C0012 with pSB1A3).
2. We transformed the linkage products into E.coli. Then we extracted the plasmids to test their qualities.
3. We transformed, extracted, and tested (BBa_K081005 and BBa_C0012 with pSB1A3).
4. We sent some of our linkage products to sequence them.
5. We extracted the BBa_B0015 and BBa_J0465
6. We cleaved the BBa_B0015 (E,X), BBa_K084012 PLUS BBa_B0015 (E, S), BBa_K084012 (E, S), BBa_K081005 (S,P), and BBa_C1002 (X,P) and we did the gel purification.
7. We amplified the BBa_I761014 by PCR.
Concentration
BBa_J0465
123.6/123.2
40/48/34
Week 5 8.11-8.17
1. We linked the BBa_K081005 and BBa_C1002; BBa_I13458 PLUS BBa_K206000 and PCR products (Traditional Assembly); BBa_K084012 and BBa_B0015; BBa_K081005 and BBa_C1002 (3A Assembly) and transformed them.
2. We extracted the plasmids and did the linkage again (3A assembly).
3. We cleaved the BBa_K084012 (E, S) (Named Cut 1), BBa_B0015 (X, P) (Cut2), BBa_I13458 PLUS BBa_K206000 (E, S) (Cut3), PCR products (X, P) (Cut4), BBa_K81005 (E, S) (Cut5), BBa_C1002 (X, P) (Cut6).
We linked the Cut1 and Cut2 (Named Lia1); Cut3 and Cut4 (Lia 2 and 3); Cut5 and Cut6 (Lia 4).
1.
Week 6 8.18-8.24
1. We cleaved the BBa_K081005 (E, S) and BBa_C1002 (X, P) plasmids.
2. We tested the lia 6-1-1 (X, P), lia 7-1-1 (X, P), lia 7-1-2 (X, P), lia 8-2-1 (E, S), and lia 8-2-2 (E, S) by cleavage and electrophoresis.
3. We did the gel purification for all the cleavage products.
4. We cleaved the BBa_K081005 (E, S), lia 6-1-4 (X, P), lia 6-1-1 (X, P), lia 6-1-2 (X, P), lia 6 -1-5 (X, P), lia 4-2-2 (E, S) and control group (E, S). Also, we prepared the linear backbones (pSB1T3 and pSB1C3) by cleavage.
5. We linked the CUT7 and CUT8 with pSB1T3 (Named NR 1), CUT3 and CUT4 with pSB1C3, pSB1T3, and pSB1K3, BBa_K081005 and CUT6 with pSB1T3 (NR 2).
6. We transformed the linkage products and extracted the BBa_I761014 and BBa_K084012 plasmids.
7. We cleaved the BBa_I761014 and BBa_C1002 with Xba I and Pst I. And we tested them by electrophoresis.
8. We amplified BBa_I761014 by PCR.
The concentration of plasmids
Concentration (ng/3 μl)
40
BBa_C0012
24
1. We tested the quality of our PCR products by electrophoresis.
2. We extracted the NR1-1, NR1-2, NR1-3, NR1-4, NR1-5, NR1-6, NR2-1, NR2-2, NR2-3, NR2-4, NR2-5, and NR2-6.
3. We tested BBa_B0015 (X, P), BBa_I13458 PLUS BBa_K206000 (E, S), BBa_C0012 (S, P), BBa_K084012 (X, P), NR1-1 (X, P), NR1-2 (X, P), NR2-1 (E, S), NR2-2 (E, S) by cleavage.
4. We cleaved NR2-3, NR2-5, NR2-6 with EcoR I and Spe I.
5. We linked the BBa_I13458 PLUS BBa_K20600 and our PCR products with pSB1K3 and pSB1C3.
6. We transformed the linkage products.
7. We tested the NR1-3, NR1-4 (E, S) and NR2-1/NR2-6 (X, P).
8. We amplified BBa_I761014 by PCR and did the PCR purification
9. We extracted NR3 and BBa_I761014 and tested them by electrophoresis and cleavage.
10. We linked BBa_I13458 PLUS BBa_K206000 and NP2 with pSB1T3 and pSB1C3.
11. We linked BBa_I13458 PLUS BBa_K206000 and BBa_I761014 with pSB1T3, BBa_0030 and BBa_C0076 with pSB1A3 and pSB1T3.
NR1-1
95.5
NR1-2
127
NR1-3
105
NR1-4
128
NR1-5
NR1-6
110
NR2-1
112
NR2-2
138
NR2-3
135
NR2-4
NR2-5
102
NR2-6
The electrophoresis results are shown in Figure 6.
Figure 6. A. the results of cleaved plasmids and PCR products. B. and C. the results of linkage products.
Sichuan university
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