"
Team:Nanjing China Bio/notebook8
From 2012.igem.org
8.15(B)
After 15 days of holiday, we returned to our experiments full of spirit and energy. In the next period, we divided our team into two parts: one in charge of extracting plasmids from bacteria strains delivered from iGEM and the following enzyme digestion and ligation with target reporting genes; the other in charge of getting anaerobic promoters we searched out from documents using PCR of bacteria strains and the following ligation with target genes.
Apart from the tasks above, we started learning the method of electric transformation, which we never got in touch with before and therefore is challenging for us.
Our team leader further emphasized some notes needed attention:
1. Mixing the agents before using them
Make sure all the agents are mixed before used. Especially for those needed to be preserved frozen, mix them up after melting. It's essential to build this awareness.
2. Operation at low temperature
Operating at low temperature can avoid non-specific annealing, therefore can avoid non-specific amplification in PCR. Apart from PCR, when operating other experiments concerning any elements that can be easily inactivated, we should have the awareness of operating at low temperature.
3. Holding the tubes in a correct way
Make sure the posture is correct when holding the tubes so that the fingers would not block the line of sight and we can watch the process of adding the liquid better.
4. Avoiding of pollution when covering the lid of PCR or centrifuge tubes
We should build an awareness of no enzyme when doing molecular experiments. Prevent contact with the inner side of the lids when covering them.
5. Keeping experiment records
We should keep the habit of recording each experiment we did.
8.21(all)
The main point of this meeting is the construction of our wiki website. After browsing the wiki websites of the teams of past iGEM competitions, we put forward different ideas about our own wiki. It came to all of us that, as the first team of Nanjing University to take part in the competition, our wiki should reflect the characteristic of our university. Meanwhile, some teammates proposed the suggestion of Chinese style. Finally, we drew the conclusion that our wiki will use Chinese painting as the background. Jingyun and Bei will take charge of the construction of our website.
After further discussion, we decided to divide our website in the following parts:
Home
About us (including our team, members, the university and the official profile of iGEM)
Projects (including our background, the methods and experiments, the protocol and the future direction)
Results (including the parts we submitted and our attribution for the iGEM)
Journal (including the meetings and the experiments)
Human practice
Safety
Above are all our initial plan, and there might be some adjustments later on.
8.26(all)
This is our last day of summer vacation, and we had this meeting to conclude our work in the summer.
For the part of the experiment, we searched out the target anaerobic promoters from documents and iGEM official website, and extracted the plasmids we wanted from bacteria strains or through strains PCR. However, we did have some problems during the experiments. The main problems among all are:
1. unsuccessful electric transformation,
2. failure in separating PCR products and primer dimers when extracting plasmids,
3. troubles like low recycle rate of gel, mistakes in electrophoresis spotting, and no separating bands in electrophoresis that repeatedly occurred during the process.
For the wiki part, we have outlined the structure of the website but have difficulty in linking to iGEM official website. Thanks to our teammates' careful records on experiments, the contents of the wiki are well prepared.
School days will begin tomorrow, and we will not be able to continue our experiments like we did in the vacation. Therefore, we rescheduled our plan for the experiments. Because our laboratory is in Gulou Campus, experiments in weekdays are in the charge of the teammates living in tGulou Campus, while teammates living in Xianlin Campus will help with the experiments in the weekends to ensure the process of the experiments. Meanwhile, teammates in Xianlin Campus will prepare for the contents for our wiki.
Experiment of A
2012.8.16
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR and add DpnⅠ(1 ul) directly to the pcr product(100 ul).
2. Purify the PCR DNA with alcohol precipitation and elute in 16L dd water.
3. Store at -80℃.
2012.8.17
1. Prepare the competent cells for electroporation. (The final concentrations of the arabinose were 10M; the inducible time is 1 hour)
2012.8.18
1. Introduce 8-10 ul DNA to competent cells by using electroporation.
2012.8.19
1. No colony was found on the plate with 50g/ml kn.
2012.8.20
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR and add DpnⅠ(1 ul) directly to the pcr product(100 ul).
2. Purify the PCR DNA with alcohol precipitation and elute in 16L dd water.
3. Store at -80℃.
2012.8.21
1. Prepare the competent cells for electroporation. (The final concentration of the arabinose was 10M; the inducible time is 1 hour)
2012.8.22
1. Introduce 8-10 ul DNA to competent cells by using electroporation.
2012.8.23
1. 5 colonies were found on the plate with 50g/ml kn.
2012.8.24
1. Pick the 5 colonies from the LB-kan plate and resuspend in10 ul of water. Do colony PCR. All of them were proved to be positive.
2012.8.25
1. Take 2 ul of the resuspended and spot it on LB-kn and incubate plate at 43C to ensure loss of PKD46.
2012.8.26
1. Verify the lost of PKD46 in the mutant strain.
Experiment of B
8.16
1. Preparation of competence VNP (take the bacteria directly without adjusting bacterial colonies).
2. Electroporation of VNP: take primers 2μl of Plate 1 1A and Plate 2 2J separately to transform.
3. PCR of bacterial colonies: two promoters, 3 derivants of one strain
Number DH5α JM109 BL27
PahdE 1 3 5
PhdeD 2 4 6
The primers contain the prefix and suffix which iGEM demands and we bring the prefix and suffix to the both ends of the promoter through PCR.
8.17
The transformation in 8.16 didn't grow any colonies.
1. Preparation of competence VNP (without adjusting bacterial colonies).
2. Cultivation of bacteria supported by iGEM committee: K239006, K376003(A), K258005, K376003(C), K376004, K387009, and K387003.
3. Electroporation of VNP: transformed 4 plasmids in total.
4. Recycle of two promoters using electrophoresis, and double digestion(30μl，E，P).
Result: the PCR of PhdeD is not satisfied.
5. Repeat the PCR of PhdeD(2, 4, 6)
Note: change the annealing time of PCR instrument to 15 s.
8.18
1. Plasmids extraction: K239005, K239011, K376006, J61043, K283025, K376003(A), K227007, K258005, K239006, K404106, K387009, K387003, K376003(C), and K376004.
2. Preparation of competence VNP
3. AGE of the PCR product yesterday; the result is not good.
8.19
1. Cultivate 4 bacterial colonies grown out of transformed VNP on 17th, and extract the plasmids to electrophorese. There appeared 3 bands, turned out a failure.
2. Make AMP+ LB solid medium.
3. Repeat the PCR of bacterial colonies in 8.16; finally, we got another group of products except for unsatisfied result of 2' and 5'.
8.20
Subculture recovered strains of VNP and TOP10 (For preparation of competence cells).
8.21
Recover strains of K239011 and K283005.
8.22
1. Help select VNP and monoclonal bacteria of Top 10 electroporation.
2. Plasmids extraction of K239011 and K283005(recovered yesterday).
3. Recover strains of (P5-21O) and (P5-23A).
8.23
1. Passage of plasmids and monoclonal strains produced in 22nd.
2. Extraction of plasmids and identification by AGE.
Experimental result: Tailing and scattered bands indicate the process of extracting plasmids might be wrong.
8.25
Extract plasmids again with the help of other members, and AGE proves successful transformation of competence bacteria using CaCl2.
8.26
1. Passage of 2 strains containing promoter mNark, 1 strain containing promoter ProssA, and 5
2. Electrophoresis of the promoter above.
3. Double digestion of 4', 5, 5 and 6 using EcoRl and Pstl.
Experimental results: Only one band of 8kb appearing indicates that VNP had been polluted.
Note: Enzyme digestion and electrophoresis identification of 4',5,5 and 6 are in the charge of other members.
8.30, 8.31
1. Electroporation of plasmid P1-1A with VNP(2011), VNP(2012), and TOP10 as comparison.
2. Colony PCR of 7 anaerobic promoters.
3. Preparation of new nutrient medium.
After 15 days of holiday, we returned to our experiments full of spirit and energy. In the next period, we divided our team into two parts: one in charge of extracting plasmids from bacteria strains delivered from iGEM and the following enzyme digestion and ligation with target reporting genes; the other in charge of getting anaerobic promoters we searched out from documents using PCR of bacteria strains and the following ligation with target genes.
Apart from the tasks above, we started learning the method of electric transformation, which we never got in touch with before and therefore is challenging for us.
Our team leader further emphasized some notes needed attention:
1. Mixing the agents before using them
Make sure all the agents are mixed before used. Especially for those needed to be preserved frozen, mix them up after melting. It's essential to build this awareness.
2. Operation at low temperature
Operating at low temperature can avoid non-specific annealing, therefore can avoid non-specific amplification in PCR. Apart from PCR, when operating other experiments concerning any elements that can be easily inactivated, we should have the awareness of operating at low temperature.
3. Holding the tubes in a correct way
Make sure the posture is correct when holding the tubes so that the fingers would not block the line of sight and we can watch the process of adding the liquid better.
4. Avoiding of pollution when covering the lid of PCR or centrifuge tubes
We should build an awareness of no enzyme when doing molecular experiments. Prevent contact with the inner side of the lids when covering them.
5. Keeping experiment records
We should keep the habit of recording each experiment we did.
8.21(all)
The main point of this meeting is the construction of our wiki website. After browsing the wiki websites of the teams of past iGEM competitions, we put forward different ideas about our own wiki. It came to all of us that, as the first team of Nanjing University to take part in the competition, our wiki should reflect the characteristic of our university. Meanwhile, some teammates proposed the suggestion of Chinese style. Finally, we drew the conclusion that our wiki will use Chinese painting as the background. Jingyun and Bei will take charge of the construction of our website.
After further discussion, we decided to divide our website in the following parts:
Home
About us (including our team, members, the university and the official profile of iGEM)
Projects (including our background, the methods and experiments, the protocol and the future direction)
Results (including the parts we submitted and our attribution for the iGEM)
Journal (including the meetings and the experiments)
Human practice
Safety
Above are all our initial plan, and there might be some adjustments later on.
8.26(all)
This is our last day of summer vacation, and we had this meeting to conclude our work in the summer.
For the part of the experiment, we searched out the target anaerobic promoters from documents and iGEM official website, and extracted the plasmids we wanted from bacteria strains or through strains PCR. However, we did have some problems during the experiments. The main problems among all are:
1. unsuccessful electric transformation,
2. failure in separating PCR products and primer dimers when extracting plasmids,
3. troubles like low recycle rate of gel, mistakes in electrophoresis spotting, and no separating bands in electrophoresis that repeatedly occurred during the process.
For the wiki part, we have outlined the structure of the website but have difficulty in linking to iGEM official website. Thanks to our teammates' careful records on experiments, the contents of the wiki are well prepared.
School days will begin tomorrow, and we will not be able to continue our experiments like we did in the vacation. Therefore, we rescheduled our plan for the experiments. Because our laboratory is in Gulou Campus, experiments in weekdays are in the charge of the teammates living in tGulou Campus, while teammates living in Xianlin Campus will help with the experiments in the weekends to ensure the process of the experiments. Meanwhile, teammates in Xianlin Campus will prepare for the contents for our wiki.
Experiment of A
2012.8.16
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR and add DpnⅠ(1 ul) directly to the pcr product(100 ul).
2. Purify the PCR DNA with alcohol precipitation and elute in 16L dd water.
3. Store at -80℃.
2012.8.17
1. Prepare the competent cells for electroporation. (The final concentrations of the arabinose were 10M; the inducible time is 1 hour)
2012.8.18
1. Introduce 8-10 ul DNA to competent cells by using electroporation.
2012.8.19
1. No colony was found on the plate with 50g/ml kn.
2012.8.20
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR and add DpnⅠ(1 ul) directly to the pcr product(100 ul).
2. Purify the PCR DNA with alcohol precipitation and elute in 16L dd water.
3. Store at -80℃.
2012.8.21
1. Prepare the competent cells for electroporation. (The final concentration of the arabinose was 10M; the inducible time is 1 hour)
2012.8.22
1. Introduce 8-10 ul DNA to competent cells by using electroporation.
2012.8.23
1. 5 colonies were found on the plate with 50g/ml kn.
2012.8.24
1. Pick the 5 colonies from the LB-kan plate and resuspend in10 ul of water. Do colony PCR. All of them were proved to be positive.
2012.8.25
1. Take 2 ul of the resuspended and spot it on LB-kn and incubate plate at 43C to ensure loss of PKD46.
2012.8.26
1. Verify the lost of PKD46 in the mutant strain.
Experiment of B
8.16
1. Preparation of competence VNP (take the bacteria directly without adjusting bacterial colonies).
2. Electroporation of VNP: take primers 2μl of Plate 1 1A and Plate 2 2J separately to transform.
3. PCR of bacterial colonies: two promoters, 3 derivants of one strain
Number DH5α JM109 BL27
PahdE 1 3 5
PhdeD 2 4 6
The primers contain the prefix and suffix which iGEM demands and we bring the prefix and suffix to the both ends of the promoter through PCR.
8.17
The transformation in 8.16 didn't grow any colonies.
1. Preparation of competence VNP (without adjusting bacterial colonies).
2. Cultivation of bacteria supported by iGEM committee: K239006, K376003(A), K258005, K376003(C), K376004, K387009, and K387003.
3. Electroporation of VNP: transformed 4 plasmids in total.
4. Recycle of two promoters using electrophoresis, and double digestion(30μl，E，P).
Result: the PCR of PhdeD is not satisfied.
5. Repeat the PCR of PhdeD(2, 4, 6)
Note: change the annealing time of PCR instrument to 15 s.
8.18
1. Plasmids extraction: K239005, K239011, K376006, J61043, K283025, K376003(A), K227007, K258005, K239006, K404106, K387009, K387003, K376003(C), and K376004.
2. Preparation of competence VNP
3. AGE of the PCR product yesterday; the result is not good.
8.19
1. Cultivate 4 bacterial colonies grown out of transformed VNP on 17th, and extract the plasmids to electrophorese. There appeared 3 bands, turned out a failure.
2. Make AMP+ LB solid medium.
3. Repeat the PCR of bacterial colonies in 8.16; finally, we got another group of products except for unsatisfied result of 2' and 5'.
8.20
Subculture recovered strains of VNP and TOP10 (For preparation of competence cells).
8.21
Recover strains of K239011 and K283005.
8.22
1. Help select VNP and monoclonal bacteria of Top 10 electroporation.
2. Plasmids extraction of K239011 and K283005(recovered yesterday).
3. Recover strains of (P5-21O) and (P5-23A).
8.23
1. Passage of plasmids and monoclonal strains produced in 22nd.
2. Extraction of plasmids and identification by AGE.
Experimental result: Tailing and scattered bands indicate the process of extracting plasmids might be wrong.
8.25
Extract plasmids again with the help of other members, and AGE proves successful transformation of competence bacteria using CaCl2.
8.26
1. Passage of 2 strains containing promoter mNark, 1 strain containing promoter ProssA, and 5
2. Electrophoresis of the promoter above.
3. Double digestion of 4', 5, 5 and 6 using EcoRl and Pstl.
Experimental results: Only one band of 8kb appearing indicates that VNP had been polluted.
Note: Enzyme digestion and electrophoresis identification of 4',5,5 and 6 are in the charge of other members.
8.30, 8.31
1. Electroporation of plasmid P1-1A with VNP(2011), VNP(2012), and TOP10 as comparison.
2. Colony PCR of 7 anaerobic promoters.
3. Preparation of new nutrient medium.
