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 7.20-7.26
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.
Week 2 7.27-8.03
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
The 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 plasmids
Week 3 8.04-8.10
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.
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 8.11-8.17
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.
Results
The 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 8.18-8.24
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.
Results
The electrophoresis results are shown in Figure 4.
Figure 4. A the gel purification results; B. the LII3 results.
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