"
Team:Nanjing China Bio/notebook9
From 2012.igem.org
9.1(all)
This is our first meeting since the beginning of the term. What's different about this meeting is that there are 3 more iGEM teams of Nanjing taking part in our meeting this time.
We talked about the condition of each team, including the work each team is working on, the facing problems, and the details about the competition in HKUST, as well as the conditions of each laboratory. Meanwhile, we decided to have 2 teach-ins in Nanjing University and Southeast University (where the 4 teams are from) in the middle of September to promote iGEM and to attract more students into this competition. After the meeting, the team leaders of the other 3 teams visited our laboratory, and they showed great appreciation for our experiments.
When the other teams left, we further concluded our work in the first week of this term. Though we met some problems during the construction of our wiki and during the process of experiments, we feel satisfied for the results we got.
We are still working on our future work. We hope to perform 2 teach-ins in Nanjing University and Southeast University and a scientific teach-in in Jinling High School in the middle of September.
9.3(A)
Today we refer to Dr. Shi from Arizona State University. He gave us the following suggestions.
1. Substitue the LB with SOB and SOC for electroporation.
2. Add arabinose when OD reaches 0.1, so the final concentration is 10mM and shake it for 4 h.
3. Precipitate the PCR with alcohol, resuspend DNA pellet in 16 ul H2O, and introduce 8-10 ul DNA to competent cells.
4. Add 500 ul SOC to grow the transformants at 37℃ for 1 h, plate 150 ul culture X2 to Cm-LB plates, and leave the rest culture on your bench. Plate them to other two Cm-LB plates next day.
We hope that we will have a better result with his advice.
9.8(B)
The key point of our meeting today is to summarize our process of the experiments. On the whole, we have a very good start at the beginning of this term. However, for many different kinds of reasons, especially the failure in separating PCR products and primer dimers when extracting plasmids, and the troubles like low recycle rate of gel, mistakes in electrophoresis like no separating bands were repeatedly spotted during the process.
However, we should feel grateful that we finally succeeded in the electroporation yesterday. It's our first success after 6 failures since last month.
As for anaerobic promoters, we finally decided the following 18 ones, and they are K387003, K404106, K227007, K376003, K239005, K239006, K376003, K288005, J61043, K387009, PadhE, PgadA, PgadE, PhdeA, PftnA, PldhA. Among these promoters, half of them are from iGEM committee, while the others are found from our references.
As for the report genes, we initially decided to use both GFP and luciferase as our report genes. However, as we have not much time to finish both of them for 18 anaerobic promoters, we have to give up GFP.
9.13(all)
Today, we want to talk about the process of our two groups of experiments.
On the whole, we have good results for both of the experiments. As the experiments of A began in June and had an early start, they seem to be more successful and have already been in the final stage. However, when it comes to our experiments of B, we have all kinds of problems, staying in a cycle period of different failures for a long time. Anyway, we are moving forward.
As for the wiki, we made some changes for it, deleting some useless parts. Besides, in order to better reflect our Chinese style, we also have some changes for our logo.
As the competition is approaching, we have more trivial things to do. Apart from the experiments, we need to prepare for other kinds of materials. We have a new arrangement for them and hope everyone can try his/her best for our final work.
9.21(all)
We are very glad that both of our experiments have had a good result now. We have worked very hard for today for over 4 months and now our efforts paid off. To celebrate our success, we go on a hiking together and feel really good about ourselves. Although we still have a lot of work to do, we have the confidence for our final competition.
Experiment of A
2012.9.3-7
Culture the B16F10 in Dulbecco's modified Eagle's medium with 10% fetal bovine serum by the routine methods.
Purchase the 6 to 8 week-old female C57BL/6 mice from Yangzhou University.
2012.9.8
These mice were implanted subcutaneously on the mid-right side with 2*105 B16F10 in 0.1ML PBS.
2012.9.15
When size of the tumor reached 500mm3,the 6-8 week-old female C57BL/6 mice were injected i.p with 0.1 ML PBS containing 104 bacteria.
2012.9.18
Isolation and titration of bacteria from the tissues were performed as described. Infected mice were euthanized and these tissues were removed aseptically and homogenized with PBS. S erially diluted homogenates were spread onto modified LB agar plates and incubated at 37℃ for 24 h.
2012.9.19
Count the bacteria on the plate (colony-forming unit [cfu] ⁄ g tissue).
2012.9.20
Use the origin software to analyze the statistics.
Experiment of B
9.1
1. 2 bacterial colonies appeared in TOP 10 with transformation of P1-1A after 18 hours. Pick monoclonal colony to cultivate.
2. Gel recycle of PCR products.
Note: 7 anaerobic porters are hde, gadE, gadA, ftnA, pfrA, ldhA, and arcA.
Result: no separating bands.
3. Electric formation of P5-21O, P5-23A, K239011, and K283025 into VNP-2011 and VNP-2012, 2 tubes for each plasmid and bacteria.
9.2
Unsuccessful transformation of yesterday's experiment.
9.3
1. Transformation of the following plasmids recombined with Luciferase with CaCl2 for plasmid amplification: K387003, K404106, K227007, K376003, K239005, K239006, K376003, K258005, and J61043.
2. Extraction and electrophoresis of P5-21O, P5-23A, K239011, and K283025.
Result: normal.
3. Extraction and electrophoresis of the 2 bacterial plasmids of TOP 10 on Sept. 1st.
Result: no bands appeared.
9.4
1. Clones of 9 plasmids heat shocked on 3rd grew out. Pick 2 clones for each to cultivate.
2. Recovery of VNP (2011 and 2012).
9.5
1. Extraction and electrophoresis of 9 plasmids cultivated on 4th.
Result: One light band of 2000bp and on dark band of 3000bp appeared. As the length of recombined plasmids is close to 3700bp and the length of the original plasmid is about 2000bp, we suspect that the enzyme digestion is not complete. Cut off the dark band and recycle the gel to re-transformation.
2. Inoculation of VNP recovered on 4th into new nutrient medium. Shake the bacteria until logarithmic phase and prepare competence bacteria.
3. Electroporation of some plasmids from one predecessor.
Note: the condition of the transformation is changed to 500Ω，1.8/2.5kV.
9.6
1. Colony PCR of 7 anaerobic promoters and recycle them with electrophoresis.
Note: PCR products can be separated under 135V in 10-15 min.
2. Preparation of solid medium, 19A+, 14C+.
3. Prepare 10 tubes of competence VNP, 5 cyropreserved, the other 5 transformed with the following 5 plasmids: P1-1A, K239011, K283025, P5-21O, and P5-23A.
4. Clones of VNP transformed with plasmids from the predecessor grew out. Pick monoclonal bacteria to cultivate.
5. Recovery of TOP 10.
9.7
No clones of 5 plasmids transformed on 6th grew out, possibly resulting from not enough washing times.
1. Extraction of plasmids cultivated on 6th showed 3 distinct bands of linear, open-loop, and super-helix types, indicating successful transformation.
2. Preparation of competence TOP 10. Recycle of recombined plasmids recycled on 5th and transformation into TOP 10.
3. Preparation of competence VNP and electroporation of the following plasmids: P1-1A, K283025, and K239011.
9.8
Some clones of the three plasmids transformed on 7th grew out.
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K387003, K404106, J61043
2. Colony PCR of the following promoters: PadhE, PgadA, PgadE
3. Electrophoresis of the promoters above to purify them
9.10
1. Extraction of the recombined plasmids: K387003, K404106, J61043
2. Electrophoresis of the digestion product yesterday to acquire the plasmid backbones with luciferase gene
3. Double digestion of PadhE, PgadA, PgadE using EcoRⅠand SpeⅠ
4. Electroporation of K387003, K404106, J61043 into VNP
9.11
Some clones of the three plasmids transformed on 10th grew out
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K227007, K376003
2. Recovery bacteria containing the following plasmid: K387009
3. Electrophoresis of the digestion product yesterday to acquire the anaerobic promoters
4. Ligate the plasmid backbones with anaerobic promoters
9.13
1. Extraction of the recombined plasmids: K227007, K376003
2. Extraction of the plasmid: K387009
3. Double digestion of K387009 using EcoRⅠand XbaⅠ
4. Colony PCR of the following promoters: PhdeA, PftnA, PldhA
5. Electrophoresis of the promoters above to purify them
6. Electroporation of K227007, K376003 and PadhE, PgadA, PgadE (with luciferase gene) into VNP
9.14
Some clones of the five plasmids transformed on 13th grew out
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K239005, K239006
2. Electrophoresis of the digestion product yesterday to acquire the plasmid backbones with luciferase gene
3. Double digestion of PhdeA, PftnA, PldhA using EcoRⅠand SpeⅠ
9.17
1. Extraction of the recombined plasmids: K239005, K239006
2. Electrophoresis of the digestion product two days ago to acquire the anaerobic promoters
3. Ligate the plasmid backbones with anaerobic promoters
4. Electroporation of K239005, K239006 into VNP
9.18
Some clones of the two plasmids transformed on 17th grew out
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K376003, K288005
2. Electroporation of PhdeA, PftnA, PldhA (with luciferase gene) into VNP
9.19
Some clones of the three plasmids transformed on 18th grew out
1. Extraction of the recombined plasmids: K376003, K288005
2. Electroporation of K376003, K288005 into VNP
9.20
Some clones of the two plasmids transformed on 19th grew out
This is our first meeting since the beginning of the term. What's different about this meeting is that there are 3 more iGEM teams of Nanjing taking part in our meeting this time.
We talked about the condition of each team, including the work each team is working on, the facing problems, and the details about the competition in HKUST, as well as the conditions of each laboratory. Meanwhile, we decided to have 2 teach-ins in Nanjing University and Southeast University (where the 4 teams are from) in the middle of September to promote iGEM and to attract more students into this competition. After the meeting, the team leaders of the other 3 teams visited our laboratory, and they showed great appreciation for our experiments.
When the other teams left, we further concluded our work in the first week of this term. Though we met some problems during the construction of our wiki and during the process of experiments, we feel satisfied for the results we got.
We are still working on our future work. We hope to perform 2 teach-ins in Nanjing University and Southeast University and a scientific teach-in in Jinling High School in the middle of September.
9.3(A)
Today we refer to Dr. Shi from Arizona State University. He gave us the following suggestions.
1. Substitue the LB with SOB and SOC for electroporation.
2. Add arabinose when OD reaches 0.1, so the final concentration is 10mM and shake it for 4 h.
3. Precipitate the PCR with alcohol, resuspend DNA pellet in 16 ul H2O, and introduce 8-10 ul DNA to competent cells.
4. Add 500 ul SOC to grow the transformants at 37℃ for 1 h, plate 150 ul culture X2 to Cm-LB plates, and leave the rest culture on your bench. Plate them to other two Cm-LB plates next day.
We hope that we will have a better result with his advice.
9.8(B)
The key point of our meeting today is to summarize our process of the experiments. On the whole, we have a very good start at the beginning of this term. However, for many different kinds of reasons, especially the failure in separating PCR products and primer dimers when extracting plasmids, and the troubles like low recycle rate of gel, mistakes in electrophoresis like no separating bands were repeatedly spotted during the process.
However, we should feel grateful that we finally succeeded in the electroporation yesterday. It's our first success after 6 failures since last month.
As for anaerobic promoters, we finally decided the following 18 ones, and they are K387003, K404106, K227007, K376003, K239005, K239006, K376003, K288005, J61043, K387009, PadhE, PgadA, PgadE, PhdeA, PftnA, PldhA. Among these promoters, half of them are from iGEM committee, while the others are found from our references.
As for the report genes, we initially decided to use both GFP and luciferase as our report genes. However, as we have not much time to finish both of them for 18 anaerobic promoters, we have to give up GFP.
9.13(all)
Today, we want to talk about the process of our two groups of experiments.
On the whole, we have good results for both of the experiments. As the experiments of A began in June and had an early start, they seem to be more successful and have already been in the final stage. However, when it comes to our experiments of B, we have all kinds of problems, staying in a cycle period of different failures for a long time. Anyway, we are moving forward.
As for the wiki, we made some changes for it, deleting some useless parts. Besides, in order to better reflect our Chinese style, we also have some changes for our logo.
As the competition is approaching, we have more trivial things to do. Apart from the experiments, we need to prepare for other kinds of materials. We have a new arrangement for them and hope everyone can try his/her best for our final work.
9.21(all)
We are very glad that both of our experiments have had a good result now. We have worked very hard for today for over 4 months and now our efforts paid off. To celebrate our success, we go on a hiking together and feel really good about ourselves. Although we still have a lot of work to do, we have the confidence for our final competition.
Experiment of A
2012.9.3-7
Culture the B16F10 in Dulbecco's modified Eagle's medium with 10% fetal bovine serum by the routine methods.
Purchase the 6 to 8 week-old female C57BL/6 mice from Yangzhou University.
2012.9.8
These mice were implanted subcutaneously on the mid-right side with 2*105 B16F10 in 0.1ML PBS.
2012.9.15
When size of the tumor reached 500mm3,the 6-8 week-old female C57BL/6 mice were injected i.p with 0.1 ML PBS containing 104 bacteria.
2012.9.18
Isolation and titration of bacteria from the tissues were performed as described. Infected mice were euthanized and these tissues were removed aseptically and homogenized with PBS. S erially diluted homogenates were spread onto modified LB agar plates and incubated at 37℃ for 24 h.
2012.9.19
Count the bacteria on the plate (colony-forming unit [cfu] ⁄ g tissue).
2012.9.20
Use the origin software to analyze the statistics.
Experiment of B
9.1
1. 2 bacterial colonies appeared in TOP 10 with transformation of P1-1A after 18 hours. Pick monoclonal colony to cultivate.
2. Gel recycle of PCR products.
Note: 7 anaerobic porters are hde, gadE, gadA, ftnA, pfrA, ldhA, and arcA.
Result: no separating bands.
3. Electric formation of P5-21O, P5-23A, K239011, and K283025 into VNP-2011 and VNP-2012, 2 tubes for each plasmid and bacteria.
9.2
Unsuccessful transformation of yesterday's experiment.
9.3
1. Transformation of the following plasmids recombined with Luciferase with CaCl2 for plasmid amplification: K387003, K404106, K227007, K376003, K239005, K239006, K376003, K258005, and J61043.
2. Extraction and electrophoresis of P5-21O, P5-23A, K239011, and K283025.
Result: normal.
3. Extraction and electrophoresis of the 2 bacterial plasmids of TOP 10 on Sept. 1st.
Result: no bands appeared.
9.4
1. Clones of 9 plasmids heat shocked on 3rd grew out. Pick 2 clones for each to cultivate.
2. Recovery of VNP (2011 and 2012).
9.5
1. Extraction and electrophoresis of 9 plasmids cultivated on 4th.
Result: One light band of 2000bp and on dark band of 3000bp appeared. As the length of recombined plasmids is close to 3700bp and the length of the original plasmid is about 2000bp, we suspect that the enzyme digestion is not complete. Cut off the dark band and recycle the gel to re-transformation.
2. Inoculation of VNP recovered on 4th into new nutrient medium. Shake the bacteria until logarithmic phase and prepare competence bacteria.
3. Electroporation of some plasmids from one predecessor.
Note: the condition of the transformation is changed to 500Ω，1.8/2.5kV.
9.6
1. Colony PCR of 7 anaerobic promoters and recycle them with electrophoresis.
Note: PCR products can be separated under 135V in 10-15 min.
2. Preparation of solid medium, 19A+, 14C+.
3. Prepare 10 tubes of competence VNP, 5 cyropreserved, the other 5 transformed with the following 5 plasmids: P1-1A, K239011, K283025, P5-21O, and P5-23A.
4. Clones of VNP transformed with plasmids from the predecessor grew out. Pick monoclonal bacteria to cultivate.
5. Recovery of TOP 10.
9.7
No clones of 5 plasmids transformed on 6th grew out, possibly resulting from not enough washing times.
1. Extraction of plasmids cultivated on 6th showed 3 distinct bands of linear, open-loop, and super-helix types, indicating successful transformation.
2. Preparation of competence TOP 10. Recycle of recombined plasmids recycled on 5th and transformation into TOP 10.
3. Preparation of competence VNP and electroporation of the following plasmids: P1-1A, K283025, and K239011.
9.8
Some clones of the three plasmids transformed on 7th grew out.
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K387003, K404106, J61043
2. Colony PCR of the following promoters: PadhE, PgadA, PgadE
3. Electrophoresis of the promoters above to purify them
9.10
1. Extraction of the recombined plasmids: K387003, K404106, J61043
2. Electrophoresis of the digestion product yesterday to acquire the plasmid backbones with luciferase gene
3. Double digestion of PadhE, PgadA, PgadE using EcoRⅠand SpeⅠ
4. Electroporation of K387003, K404106, J61043 into VNP
9.11
Some clones of the three plasmids transformed on 10th grew out
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K227007, K376003
2. Recovery bacteria containing the following plasmid: K387009
3. Electrophoresis of the digestion product yesterday to acquire the anaerobic promoters
4. Ligate the plasmid backbones with anaerobic promoters
9.13
1. Extraction of the recombined plasmids: K227007, K376003
2. Extraction of the plasmid: K387009
3. Double digestion of K387009 using EcoRⅠand XbaⅠ
4. Colony PCR of the following promoters: PhdeA, PftnA, PldhA
5. Electrophoresis of the promoters above to purify them
6. Electroporation of K227007, K376003 and PadhE, PgadA, PgadE (with luciferase gene) into VNP
9.14
Some clones of the five plasmids transformed on 13th grew out
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K239005, K239006
2. Electrophoresis of the digestion product yesterday to acquire the plasmid backbones with luciferase gene
3. Double digestion of PhdeA, PftnA, PldhA using EcoRⅠand SpeⅠ
9.17
1. Extraction of the recombined plasmids: K239005, K239006
2. Electrophoresis of the digestion product two days ago to acquire the anaerobic promoters
3. Ligate the plasmid backbones with anaerobic promoters
4. Electroporation of K239005, K239006 into VNP
9.18
Some clones of the two plasmids transformed on 17th grew out
1. Recovery bacteria containing the following recombined plasmids (with luciferase): K376003, K288005
2. Electroporation of PhdeA, PftnA, PldhA (with luciferase gene) into VNP
9.19
Some clones of the three plasmids transformed on 18th grew out
1. Extraction of the recombined plasmids: K376003, K288005
2. Electroporation of K376003, K288005 into VNP
9.20
Some clones of the two plasmids transformed on 19th grew out
