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 Transmitter Back to top Week 1 Week 2 Week 3 Week 4 Week 5 Since our team is sorted into 4 groups, our team is focusing on the construction of the Transmitter Cell. The whole work that we should do is to build 2 gene lines which lie in the transmitter cells.In detail, we use 9 biobricks supplied by IGEM Competition totally which are RBS (BBa_B0030), DT (BBa_B0015), pLux (BBa_R0062), LuxR (BBa_C0062), LasI (BBa_K081016 and BBa_K081009), CinR (BBa_K0077), RhlR (BBa_C0171), and RhlI (BBa_C0051).Week 1 7.20-7.261. We transformed the plasmids (BBa_B0030, BBa_B0015, BBa_R0062, BBa_K081016, BBa_K081009, BBa_K0077, BBa_C0171, BBa_C0051) into DH5α E.Coli to amplify these plasmidWe extracted the plasmids (mentioned above) and used electrophoresis to test the concentration and quality of our plasmids.Results:Name Concentration (ng/3 μl) BBa_R0062 53.3/ 43.5 BBa_K081016 107.9/ 176.7 BBa_B0030 28.2,/8.96 BBa_C0171 322.6/ 387.5 BBa_K0077 193.4,/143.9 BBa_K081009 64.4,/63.1 BBa_C0077 152.7/ 156 BBa_C0062 53.29/128.7 BBa_B0015 116.2/19.5 Note: The different concentrations are from different tubes.Week 2 7.27-8.031. We transformed the BBa_C0062 and BBa_C0077 plasmids.2. We extracted the BBa_J37019, BBa_I9026, BBa_K081005, BBa_I714075, BBa_K93600 and BBa_R0077 plasmids and determined the concentration of them by electrophoresis.3. We cleaved the BBa_R0062, BBa_C0051, BBa_K0077 plasmids with EcoR I and Spe I enzyme and BBa_B0015 with Xba I and Pst I. Also, we prepared the pSB1A3 and pSB1K3 backbones with EcoR I and Pst I.4. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I.5. We linked the E11 and E12 with pSB1A3 (L11), the E21 and E22 with pSB1A3 (L21).6. We transformed all the linked parts into E.coli.7. We cleaved BBa_B0015 with Xba I and Pst I.ResultsThe concentration of plasmidsName Concentration (ng/3μl) BBa_J37019 400/403 BBa_I9026 247/140.6 BBa_R0077 134/161.2 BBa_K081005 51.2/27.6 BBa_I714075 95.7/37.2 BBa_K93600 41.6 L1 92.3/54.2 L2 88.4/60.4 Note: The different concentrations are from different tubes.2. The electrophoresis results are shown in Figure 1. Figure 1. The cleaved plasmidsWeek 3 8.04-8.101. We linked the L1 and BBa_B0015 with pSB1T3 (named F1), L2 and BBa_I9026 with pSB1T3 (F2). Then we transformed them into E.coli to amplify them and we extracted the plasmids to test their quality.2. We linked E11 and E12 with pSB1T3 (named LA1), E21 and E22 with pSB1A3 (LA2).3. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I again and tested their qualities by electrophoresis and then we did the gel purification.4. We cleaved the BBa_R0077 with Spe I and Pst I (named EP21).5. We linked EP21 and E22 with 3 different backbones (pSB1T3, pSB1A3, and pSB1C3) which are tested by electrophoresis.Results:The concentration of plasmids and linkage productName Concentration (ng/3μl) F11 Failed F21 failed E11 8.53 E12 Failed E21 24.9/7.5 E22 14/5.3 E24 18.1/17.7 Note: The different concentrations are from different tubes.2. The electrophoresis results are shown in Figure 2.Figure 2. A, C, and D the gel purification results and cleaved plasmids. B. the cleaved EP21.Week 4 8.11-8.171. We did the liquid culture of BBa_R0077 and BBa_C0077.2. We extracted the BBa_J37019, BBa_I9026 plasmids and then we cleaved both of them (BBa_J37019 with Spe I and Pst I; BBa_I9026 with Xba I and Pst I) and linked them with pSB1C3. Finally we tested the quality by transforming into E.coli.3. We linked BBa_J37019 and BBa_I9026(LI1), BBa_R0077 and BBa_C0077 with pSB1C3.4. We extracted BBa_I9026, BBa_J37019, BBa_C0077, and BBa_R0077.5. We cleaved BBa_J37019 with E, S (D1, D2) or S, P (D3, D4) and BBa_I9026 plasmids with E, S, (D5, D6) and S, P (D7, D8) enzymes. Then we did the gel purification.6. We linked the BBa_J37019 with BBa_I9026 with different types (named LI3).7. We transformed LI3 into E.coli and extracted the plasmids to test the quality.ResultsThe concentration of plasmidsName Concentration (ng/3μl) BBa_I9026 289.2/112.6 BBa_J37019 193.2/40.7 BBa_C0077 27.7/22.5/33.3 BBa_R0077 177.8/179.8/200.3 Note: The different concentrations are from different tubes.2. The electrophoresis results are shown in Figure 3.Figure A. the cleaved BBa_J37019 and BBa_I9026 plasmids. B. the cleaved linkage products. C and D is the gel purification of BBa_J37019 and BBa_I9026. E. the cleaved BBa_R0077 and BBa_C0077.F. the linkage products.Week 5 8.18-8.241. We linked the E11 and E12 with pSB1T3 by 3A assembly and E11 and E12 by traditional assembly.2. We Amplified the LII3 which was tested by cleavage and electrophoresis.3. We transformed the BBa_J37019, BBa_I9026, BBa_C0077, and L1T1.4. We cultured the LIIF in liquid and transformed it.5. We did the gel purification of BBa_J37019 and BBa_K747092.ResultsThe electrophoresis results are shown in Figure 4.Figure 4. A the gel purification results; B. the LII3 results. Sichuan university
The Notebook of
Transmitter
Since our team is sorted into 4 groups, our team is focusing on the construction of the Transmitter Cell. The whole work that we should do is to build 2 gene lines which lie in the transmitter cells.
In detail, we use 9 biobricks supplied by IGEM Competition totally which are RBS (BBa_B0030), DT (BBa_B0015), pLux (BBa_R0062), LuxR (BBa_C0062), LasI (BBa_K081016 and BBa_K081009), CinR (BBa_K0077), RhlR (BBa_C0171), and RhlI (BBa_C0051).
1. We transformed the plasmids (BBa_B0030, BBa_B0015, BBa_R0062, BBa_K081016, BBa_K081009, BBa_K0077, BBa_C0171, BBa_C0051) into DH5α E.Coli to amplify these plasmid
We extracted the plasmids (mentioned above) and used electrophoresis to test the concentration and quality of our plasmids.
Results:
Name
Concentration (ng/3 μl)
BBa_R0062
53.3/ 43.5
BBa_K081016
107.9/ 176.7
BBa_B0030
28.2,/8.96
BBa_C0171
322.6/ 387.5
BBa_K0077
193.4,/143.9
BBa_K081009
64.4,/63.1
BBa_C0077
152.7/ 156
BBa_C0062
53.29/128.7
BBa_B0015
116.2/19.5
Note: The different concentrations are from different tubes.
1. We transformed the BBa_C0062 and BBa_C0077 plasmids.
2. We extracted the BBa_J37019, BBa_I9026, BBa_K081005, BBa_I714075, BBa_K93600 and BBa_R0077 plasmids and determined the concentration of them by electrophoresis.
3. We cleaved the BBa_R0062, BBa_C0051, BBa_K0077 plasmids with EcoR I and Spe I enzyme and BBa_B0015 with Xba I and Pst I. Also, we prepared the pSB1A3 and pSB1K3 backbones with EcoR I and Pst I.
4. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I.
5. We linked the E11 and E12 with pSB1A3 (L11), the E21 and E22 with pSB1A3 (L21).
6. We transformed all the linked parts into E.coli.
7. We cleaved BBa_B0015 with Xba I and Pst I.
Results
Concentration (ng/3μl)
BBa_J37019
400/403
BBa_I9026
247/140.6
BBa_R0077
134/161.2
BBa_K081005
51.2/27.6
BBa_I714075
95.7/37.2
BBa_K93600
41.6
L1
92.3/54.2
L2
88.4/60.4
2. The electrophoresis results are shown in Figure 1.
Figure 1. The cleaved plasmids
1. We linked the L1 and BBa_B0015 with pSB1T3 (named F1), L2 and BBa_I9026 with pSB1T3 (F2). Then we transformed them into E.coli to amplify them and we extracted the plasmids to test their quality.
2. We linked E11 and E12 with pSB1T3 (named LA1), E21 and E22 with pSB1A3 (LA2).
3. We cleaved the BBa_J37019 (Named E11) and BBa_R0077 (E21) with EcoR I and Spe I, BBa_I9026 (E12), BBa_K081016 (E22) and BBa_C0077 (E24) with Xba I and Pst I again and tested their qualities by electrophoresis and then we did the gel purification.
4. We cleaved the BBa_R0077 with Spe I and Pst I (named EP21).
5. We linked EP21 and E22 with 3 different backbones (pSB1T3, pSB1A3, and pSB1C3) which are tested by electrophoresis.
F11
Failed
F21
failed
E11
8.53
E12
E21
24.9/7.5
E22
14/5.3
E24
18.1/17.7
2. The electrophoresis results are shown in Figure 2.
Figure 2. A, C, and D the gel purification results and cleaved plasmids. B. the cleaved EP21.
1. We did the liquid culture of BBa_R0077 and BBa_C0077.
2. We extracted the BBa_J37019, BBa_I9026 plasmids and then we cleaved both of them (BBa_J37019 with Spe I and Pst I; BBa_I9026 with Xba I and Pst I) and linked them with pSB1C3. Finally we tested the quality by transforming into E.coli.
3. We linked BBa_J37019 and BBa_I9026(LI1), BBa_R0077 and BBa_C0077 with pSB1C3.
4. We extracted BBa_I9026, BBa_J37019, BBa_C0077, and BBa_R0077.
5. We cleaved BBa_J37019 with E, S (D1, D2) or S, P (D3, D4) and BBa_I9026 plasmids with E, S, (D5, D6) and S, P (D7, D8) enzymes. Then we did the gel purification.
6. We linked the BBa_J37019 with BBa_I9026 with different types (named LI3).
7. We transformed LI3 into E.coli and extracted the plasmids to test the quality.
289.2/112.6
193.2/40.7
27.7/22.5/33.3
177.8/179.8/200.3
2. The electrophoresis results are shown in Figure 3.
Figure A. the cleaved BBa_J37019 and BBa_I9026 plasmids. B. the cleaved linkage products. C and D is the gel purification of BBa_J37019 and BBa_I9026. E. the cleaved BBa_R0077 and BBa_C0077.
F. the linkage products.
1. We linked the E11 and E12 with pSB1T3 by 3A assembly and E11 and E12 by traditional assembly.
2. We Amplified the LII3 which was tested by cleavage and electrophoresis.
3. We transformed the BBa_J37019, BBa_I9026, BBa_C0077, and L1T1.
4. We cultured the LIIF in liquid and transformed it.
5. We did the gel purification of BBa_J37019 and BBa_K747092.
The electrophoresis results are shown in Figure 4.
Figure 4. A the gel purification results; B. the LII3 results.
Sichuan university
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