"
Team:Nanjing China Bio/notebook7
From 2012.igem.org
7.05(B)
This is our first regular meeting after the final exam. Considering some teammates have gone home, we simply concluded our work before and afterwards.
After analyzing many documents about Salmonella, we decided to start with improveing the character of facultative anaerobic of the bacteria to enhance its property of tumor targeting.
From the documents, we realized that the reason why some bacteria are highly anaerobic is related to anaerobic promoters. The most used anaerobic promoter applied in experiments to express the target genes at present is promoter nir B. However, we found other anaerobic promoters had few differences from Pnir B based on the data. Therefore we supposed there might be better anaerobic promoters than Pnir B in terms of expressing specific target genes, and set our next goal to find newer and better anaerobic promoters.
In order to realize this goal, we start with reading the following documents that might bring us some illumination.
Ganai S, Arenas RB, Forbes NS. Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice. British Journal of Cancer 2009;101:1683-91.
Brader P,Stritzker J, Riedl CC,etal. Escherichia coli Nissle 1917 facilitates tumor detection by positron emission tomography and optical imaging. Clinical Cancer Research 2008;14:2295-302.
Weth R, Christ O, Stevanovic S,etal. Gene delivery by attenuated Salmonella typhimurium: Comparing the efficacy of helper versus cytotoxic T cell priming in tumor vaccination. Cancer Gene Therapy 2001;8:599-611.
Loeffler M, Le'Negrate G, Krajewska M, etal. Attenuated Salmonella engineered to produce human cytokine LIGHT inhibit tumor growth. Proceedings of the National Academy of Sciences of the United States of America 2007;104:12879-83.
Loeffler M, Le'Negrate G, Krajewska M, etal. Inhibition of tumor growth using Salmonella expressing Fas ligand. Journal of the National Cancer Institute 2008;100:1113-6.
7.14（B）
After all the hard work of reading documents, we searched out the following anaerobic promoters that might be better than Promoter nir B: Nark, fdeF, J6 oxygen promoter, puc promoter, VHb promoter, mNark, phTERT, and pfdhF. We hope to identify through experiments whether the property of these promoters is better than Promoter nir B when pressing the target gene we need as we expected.
Meanwhile, we found some anaerobic promoters that we think could be capable of our requirements from the iGEM official website: K239005, K239011, K376006, J61043, K283025, K376003(A), K227007, K258005, K239006, K404106, K387009, K387003, K376003(C), and K376004. We hope we could testify all these promoters after we received the plat from iGEM.
7.15(A)
Recently we failed to knock out the relevant genes of VNP20009
The final of concentration of arabinose we used was from 10mMol to 100mMol and the inducible time was from 1 to 4 hours. However we didn't get the mutants in salmonella.
Recently we read an article Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes, in this article the writer used several ways to improve efficacy of recombinant, First we should check the helper plasmid pKD46 in the first step of the method. Secondly LB agar plates containing kn were used as selective media. Initially we used 50g/ml kn. however this paper recommend that we use lower antibiotic concentrations in the selective media. Once plated, 24 hours of incubation were enough to visualize colonies. The colonies were then transferred to new LB agar plates with higher antibiotic concentrations (kn: 50μg/ml).
7.18(B)
After several days of consideration, we began to draw out our experiment plan.
Considering that the plat from iGEM and the bacteria strains and enzymes we need are not in readiness, we decided to start with the basic experiments of PCR, extraction of plasmids and recycle of plasmids. In the following days, we will perform the following experiments as practice.
1. Extraction of plasmids.
2. PCR.
3. DNA agarose gel electrophoresis.
4. DNA restriction endonuclease reaction.
5. Recycle of DNA.
As most of us are from the School of Life Science and have already had the experience of these experiments, we expect we would have a good start and get into the best condition soon.
7.30(B)
After 10 days of pre-experiments, we decided to start our experiments officially tomorrow. We have basically determined our project. Firstly, we hope to extract the plasmids from the bacteria strains from iGEM that might contain anaerobic promoters we want. And for the plasmids we searched out from documents, we decided to get them through PCR. Secondly is specific enzyme digestion at EX or SP site. Following is adding report genes into EX or SP site. The final step is to transfer the plasmids into VNP through electric transformation and cultivate the bacteria anaerobically. Then examine the expression intensity of report genes to determine the property of the anaerobic promoters and to find out the best one.
Experiments of A
2012.7.2
1. Prepare the competent cells for electroporation.
2. Introduce PKD4 into VNP20009 using electroporation procedure.
2012.7.3
1. Pick up the single colony.
2. Grow the VNP20009 harboring the PKD4 in LB +AMP at 30℃ overnight.
3. Prepare the competent cells for electroporation.
4. Introduce PKD4 into VNP20009 using electroporation procedure.
2012.7.4
1. Pick up the single colony.
2. Grow the VNP20009 harboring the PKD4 in LB +AMP at 30℃ overnight.
2012.7.5
1. The kanamycin cassette with the relevant homologous region were successfully amplified by the PCR.
2. Purify the PCR DNA and store at -80℃.
2012.7.5
1. Introduce the kanamycin cassette with the relevant homologous region into the vnp20009 with pkd4 and spread onto the plates that contain 25g/ML kn.
2012.7.6
1．No colony was found on the plate.
2012.7.10
1. The kanamycin cassette with the relevant homologous region were successfully amplified by the PCR.
2. Purify the PCR DNA and store at -80℃.
2012.7.11
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with pkd4 and spread onto the plates that contain 20g/ML kn.
2012.7.12
1．Multiple small colonies were found on the plate, however none of them were positive.
2012.7.16
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR.
2. Purify the he PCR DNA with bocai kit and elute in 30L dd water.
3. Store at -80℃.
2012.7.18
1. Prepare the competent cells for electroporation.(The final concentrations of the arabinose were 100M or 10M; the inducible time is 1, 2 , 3 and 4 hours respectively)
2012.7.19
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4 (10-1, 10-2, 10-3, 10-4, 100-1, 100-2, 100-3, 100-4).
2012.7.20
1. Multiple colonies were found on the plate with 20g/ml kn, however none of them was positive.
2012.7.24
1. Amplify the kanamycin cassette with the relevant homologous region with PCR.
2. Purify the PCR DNA with Biocolors kit and elute in 30L dd H2O.
3. Store at -80℃.
2012.7.25
1. Prepare the competent cells for electroporation.(The final concentrations of the arabinose were 100M or 10M; the inducible time is 1, 2 , 3 and 4 hours respectively.)
2012.7.26
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4 (10-1, 10-2, 10-3, 10-4, 100-1, 100-2, 100-3, 100-4).
2012.7.27
1. Multiple colonies were found on the plate with 20g/ml kn, however none of them was positive.
Experiments of B
7.20
1. PCR reaction (50μl)
2. Electrophoresis of PCR products to see if the amplification is successful
Result: success in PCR
7.21
1. Miniprep of plasmids
2. Enzyme digestion (30μl, BanH I, Hind III, overnight)
3. Preparation of competent bacteria with CaCl2
4. Recycle of gel
7.22
1. Ligation (10μl)
2. Electroporation
7.24
1. Cultivation of DH5α bacteria containing plasmid pQE30
2. Inoculation and cultivation of TOP 10 E.coli
3. Annealing of primers, forming double-strand target segments
7.25
1. Miniprep of pQE30
2. Preparation of competent TOP 10 with CaCl2
3. Double enzyme digestion of pQE30 (30μl, BamH I, Hind III, 3h)
4. Gel recycle of digestion products
5. Ligation (4h)
6. Electroporation
7.26
The petri dish is full of bacteria cultivated on 25th, and single bacteria colony could barely be seen, indicating competent bacteria are polluted of bacteria with resistant.
1. Miniprep of TOP 10 suspected to be polluted
Result: light bands appeared in the electrophoresis, showing TOP 10 have been polluted.
2. Re-inoculation of TOP 10
3. Re-ligation of products of enzyme digestion of pQE30 and annealing.
7.28
1. Miniprep of plasmids pQE30 of TOP 10 strains
2. Determination of enzyme digestion
Result: Because there are EcoR I site on both target segments and the original plasmid, two bands appeared in the electrophoresis, showing successful annealing of annealing products.
7.29
1. Preparation of competent TOP 10
2. Transformation of TOP 10 with iGEM distribution plate P1-15J
7.30
Failure in transformation of 29th, so repeat yesterday's experiment.
7.31
Success in transformation of 30th, pick monoclonal bacteria to cultivate.
This is our first regular meeting after the final exam. Considering some teammates have gone home, we simply concluded our work before and afterwards.
After analyzing many documents about Salmonella, we decided to start with improveing the character of facultative anaerobic of the bacteria to enhance its property of tumor targeting.
From the documents, we realized that the reason why some bacteria are highly anaerobic is related to anaerobic promoters. The most used anaerobic promoter applied in experiments to express the target genes at present is promoter nir B. However, we found other anaerobic promoters had few differences from Pnir B based on the data. Therefore we supposed there might be better anaerobic promoters than Pnir B in terms of expressing specific target genes, and set our next goal to find newer and better anaerobic promoters.
In order to realize this goal, we start with reading the following documents that might bring us some illumination.
Ganai S, Arenas RB, Forbes NS. Tumour-targeted delivery of TRAIL using Salmonella typhimurium enhances breast cancer survival in mice. British Journal of Cancer 2009;101:1683-91.
Brader P,Stritzker J, Riedl CC,etal. Escherichia coli Nissle 1917 facilitates tumor detection by positron emission tomography and optical imaging. Clinical Cancer Research 2008;14:2295-302.
Weth R, Christ O, Stevanovic S,etal. Gene delivery by attenuated Salmonella typhimurium: Comparing the efficacy of helper versus cytotoxic T cell priming in tumor vaccination. Cancer Gene Therapy 2001;8:599-611.
Loeffler M, Le'Negrate G, Krajewska M, etal. Attenuated Salmonella engineered to produce human cytokine LIGHT inhibit tumor growth. Proceedings of the National Academy of Sciences of the United States of America 2007;104:12879-83.
Loeffler M, Le'Negrate G, Krajewska M, etal. Inhibition of tumor growth using Salmonella expressing Fas ligand. Journal of the National Cancer Institute 2008;100:1113-6.
7.14（B）
After all the hard work of reading documents, we searched out the following anaerobic promoters that might be better than Promoter nir B: Nark, fdeF, J6 oxygen promoter, puc promoter, VHb promoter, mNark, phTERT, and pfdhF. We hope to identify through experiments whether the property of these promoters is better than Promoter nir B when pressing the target gene we need as we expected.
Meanwhile, we found some anaerobic promoters that we think could be capable of our requirements from the iGEM official website: K239005, K239011, K376006, J61043, K283025, K376003(A), K227007, K258005, K239006, K404106, K387009, K387003, K376003(C), and K376004. We hope we could testify all these promoters after we received the plat from iGEM.
7.15(A)
Recently we failed to knock out the relevant genes of VNP20009
The final of concentration of arabinose we used was from 10mMol to 100mMol and the inducible time was from 1 to 4 hours. However we didn't get the mutants in salmonella.
Recently we read an article Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes, in this article the writer used several ways to improve efficacy of recombinant, First we should check the helper plasmid pKD46 in the first step of the method. Secondly LB agar plates containing kn were used as selective media. Initially we used 50g/ml kn. however this paper recommend that we use lower antibiotic concentrations in the selective media. Once plated, 24 hours of incubation were enough to visualize colonies. The colonies were then transferred to new LB agar plates with higher antibiotic concentrations (kn: 50μg/ml).
7.18(B)
After several days of consideration, we began to draw out our experiment plan.
Considering that the plat from iGEM and the bacteria strains and enzymes we need are not in readiness, we decided to start with the basic experiments of PCR, extraction of plasmids and recycle of plasmids. In the following days, we will perform the following experiments as practice.
1. Extraction of plasmids.
2. PCR.
3. DNA agarose gel electrophoresis.
4. DNA restriction endonuclease reaction.
5. Recycle of DNA.
As most of us are from the School of Life Science and have already had the experience of these experiments, we expect we would have a good start and get into the best condition soon.
7.30(B)
After 10 days of pre-experiments, we decided to start our experiments officially tomorrow. We have basically determined our project. Firstly, we hope to extract the plasmids from the bacteria strains from iGEM that might contain anaerobic promoters we want. And for the plasmids we searched out from documents, we decided to get them through PCR. Secondly is specific enzyme digestion at EX or SP site. Following is adding report genes into EX or SP site. The final step is to transfer the plasmids into VNP through electric transformation and cultivate the bacteria anaerobically. Then examine the expression intensity of report genes to determine the property of the anaerobic promoters and to find out the best one.
Experiments of A
2012.7.2
1. Prepare the competent cells for electroporation.
2. Introduce PKD4 into VNP20009 using electroporation procedure.
2012.7.3
1. Pick up the single colony.
2. Grow the VNP20009 harboring the PKD4 in LB +AMP at 30℃ overnight.
3. Prepare the competent cells for electroporation.
4. Introduce PKD4 into VNP20009 using electroporation procedure.
2012.7.4
1. Pick up the single colony.
2. Grow the VNP20009 harboring the PKD4 in LB +AMP at 30℃ overnight.
2012.7.5
1. The kanamycin cassette with the relevant homologous region were successfully amplified by the PCR.
2. Purify the PCR DNA and store at -80℃.
2012.7.5
1. Introduce the kanamycin cassette with the relevant homologous region into the vnp20009 with pkd4 and spread onto the plates that contain 25g/ML kn.
2012.7.6
1．No colony was found on the plate.
2012.7.10
1. The kanamycin cassette with the relevant homologous region were successfully amplified by the PCR.
2. Purify the PCR DNA and store at -80℃.
2012.7.11
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with pkd4 and spread onto the plates that contain 20g/ML kn.
2012.7.12
1．Multiple small colonies were found on the plate, however none of them were positive.
2012.7.16
1. Amplify the the kanamycin cassette with the relevant homologous region with PCR.
2. Purify the he PCR DNA with bocai kit and elute in 30L dd water.
3. Store at -80℃.
2012.7.18
1. Prepare the competent cells for electroporation.(The final concentrations of the arabinose were 100M or 10M; the inducible time is 1, 2 , 3 and 4 hours respectively)
2012.7.19
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4 (10-1, 10-2, 10-3, 10-4, 100-1, 100-2, 100-3, 100-4).
2012.7.20
1. Multiple colonies were found on the plate with 20g/ml kn, however none of them was positive.
2012.7.24
1. Amplify the kanamycin cassette with the relevant homologous region with PCR.
2. Purify the PCR DNA with Biocolors kit and elute in 30L dd H2O.
3. Store at -80℃.
2012.7.25
1. Prepare the competent cells for electroporation.(The final concentrations of the arabinose were 100M or 10M; the inducible time is 1, 2 , 3 and 4 hours respectively.)
2012.7.26
1. Introduce the kanamycin cassette with the relevant homologous region into the VNP20009 with PKD4 (10-1, 10-2, 10-3, 10-4, 100-1, 100-2, 100-3, 100-4).
2012.7.27
1. Multiple colonies were found on the plate with 20g/ml kn, however none of them was positive.
Experiments of B
7.20
1. PCR reaction (50μl)
2. Electrophoresis of PCR products to see if the amplification is successful
Result: success in PCR
7.21
1. Miniprep of plasmids
2. Enzyme digestion (30μl, BanH I, Hind III, overnight)
3. Preparation of competent bacteria with CaCl2
4. Recycle of gel
7.22
1. Ligation (10μl)
2. Electroporation
7.24
1. Cultivation of DH5α bacteria containing plasmid pQE30
2. Inoculation and cultivation of TOP 10 E.coli
3. Annealing of primers, forming double-strand target segments
7.25
1. Miniprep of pQE30
2. Preparation of competent TOP 10 with CaCl2
3. Double enzyme digestion of pQE30 (30μl, BamH I, Hind III, 3h)
4. Gel recycle of digestion products
5. Ligation (4h)
6. Electroporation
7.26
The petri dish is full of bacteria cultivated on 25th, and single bacteria colony could barely be seen, indicating competent bacteria are polluted of bacteria with resistant.
1. Miniprep of TOP 10 suspected to be polluted
Result: light bands appeared in the electrophoresis, showing TOP 10 have been polluted.
2. Re-inoculation of TOP 10
3. Re-ligation of products of enzyme digestion of pQE30 and annealing.
7.28
1. Miniprep of plasmids pQE30 of TOP 10 strains
2. Determination of enzyme digestion
Result: Because there are EcoR I site on both target segments and the original plasmid, two bands appeared in the electrophoresis, showing successful annealing of annealing products.
7.29
1. Preparation of competent TOP 10
2. Transformation of TOP 10 with iGEM distribution plate P1-15J
7.30
Failure in transformation of 29th, so repeat yesterday's experiment.
7.31
Success in transformation of 30th, pick monoclonal bacteria to cultivate.
