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Team:Colombia/Notebook/Journal
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===June 20=== | ===June 20=== | ||
Today we supposed to amplify all the genes for the detection system but PxpsR (those primers haven’t arrive yet). We used Fermentas Pfu (see protocols) and the Ta for each gene was calculated taking the lower primer Tm of each couple and subtracting it 2 degrees (phcS=61°C, phcR=57°C, phcA=54°C). Nonetheless, none amplified. We are no longer sure if the primers anneal in all Ralstonia strains, we haven’t consider that before. | Today we supposed to amplify all the genes for the detection system but PxpsR (those primers haven’t arrive yet). We used Fermentas Pfu (see protocols) and the Ta for each gene was calculated taking the lower primer Tm of each couple and subtracting it 2 degrees (phcS=61°C, phcR=57°C, phcA=54°C). Nonetheless, none amplified. We are no longer sure if the primers anneal in all Ralstonia strains, we haven’t consider that before. | ||
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===June 21=== | ===June 21=== | ||
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|1L | |1L | ||
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| + | |||
| + | ===June 24=== | ||
| + | We decided to make a pilot experiment in order to standardize the conditions of phcA amplification before to start with the massive screening . We choose #37 strain randomly for the standardization. | ||
| + | |||
| + | ===June 26=== | ||
| + | In order to standardize the conditions of phcA amplification before to start with the massive screening , we determine the annealing temperature (71°C) performing an in silico PCR (FAST PCR) and used that temperature to calculate a temperature gradient (form 64 to 71), reactions were carry out with or without DMSO. The results weren’t expected, none of the temperatures amplified. | ||
| + | |||
| + | ===June 27=== | ||
| + | We perfomed phcA PCRs using a boiling of the solid culture as a source of DNA. The results remained the same. Due to results we decided to change of strains and we growth on solid 3 new accessions. | ||
| + | |||
| + | ===June 29=== | ||
Revision as of 00:23, 14 July 2012
Contents |
The Journal
Chitinase
To determine which chitinase is going to be used, we screened chitinase from 3 different species since the chitinase of different Vibrio spp. was not suitable or the strain was found (Alivibrio fischeri ES114, Alivibrio fischeri M11):
- Arabidopsis thaliana
- Colletotrichum spp.
- Trichoderma spp.
For each one we obtained accesion numbers:
- Arabidopsis thaliana: [http://www.ncbi.nlm.nih.gov/nuccore/AY099810.2 AY099810.2]
- Colletotrichum spp. : [http://www.ncbi.nlm.nih.gov/nucest/GW342409.1 GW342409.1]
- Trichoderma spp. : [http://www.ncbi.nlm.nih.gov/nucest/BM077089.2 BM077089.2]
Each one was tested using codon usage for bacteria (Translation table 11) to determine if the chitinase could be used. All of the sequences were suitable for use.
We selected Colletotrichum spp. and Trichoderma spp. sequences to design primers.
Primers
- Colletotrichum spp. :
- Trichoderma spp. :
Ralstonia solanacearum
June 8
Today we have our first meeting! We basically talk about the Ralstonia detection system and make a little research to find a proper medium to growth Ralstonia. Here is a little sketch we made.
June 12
We decided to prepare Casamino acid-Peptone-Glucose (CPG) media, which is a rich medium and we also have all the components in the lab. This is the recipe for 1L of CPG:
| Reactives | Amount (g) |
|---|---|
| Casamino acid | 1 |
| Peptone | 10 |
| Glucose | 5 |
| Agar | 15 |
June 13
We growth in solid medium a Ralstonia strain from the REVCO, it belongs to the phytopathogen bacteria strain store of the LAMFU, it will fully growth in 2 days, so we have to wait until then…everything is pretty easy so far.
June 15
We were going to extract DNA from Ralstonia tomorrow, so today we spend a lot of time preparing the solutions, but our Ralstonia strain didn’t growth, we are kind of upset. We also design the primers to amplify the promoter of xpsR! We used the sequence of ...
June 16
Today is a new day, we decided to growth a different accession of Ralstonia and see what happens.
June 18
Our Ralstonia is alive! it not longer matter that today’s holyday and we are at lab…We are finally extracting DNA tomorrow, so we inoculated 5 ml of liquid CPG with a few colonies from the solid culture and left growth ON.
June 19
Today was DNA extraction day! We used the Xam’s DNA extraction protocol(see protocols) and here it is, our Ralstonia’s DNA, we think is a little bit degraded but still amplifiable…
June 20
Today we supposed to amplify all the genes for the detection system but PxpsR (those primers haven’t arrive yet). We used Fermentas Pfu (see protocols) and the Ta for each gene was calculated taking the lower primer Tm of each couple and subtracting it 2 degrees (phcS=61°C, phcR=57°C, phcA=54°C). Nonetheless, none amplified. We are no longer sure if the primers anneal in all Ralstonia strains, we haven’t consider that before.
June 21
We performed a primer search in NCBI and confirmed our suspects … there are some base changes at the 3’ of our primers in the sequence of different strains of R. solanacearum…that will explain why it didn’t amplified a thing yesterday. The most conserved sequence appear to be PxpsR and then phcA, so, while PxpsR primers arrive, we will perform a screening of differtent Ralstonia solanacearum strains using phcA, then we will try to amplify the other genes from positive strains. Here is our decision tree.
June 22
Today we prepared the selective media for the screening; we had to make substantial variations due to a lack of compounds in our lab. This was the final composition:
| Reactives | Amount |
|---|---|
| Mannitol | 1g |
| Na2HPO4 | 3g |
| KH2PO4 | 3g |
| NH4Cl | 1g |
| MgSO4 | 0.25g |
| FeSO4 | 5mg |
| Crystal Violet | 3mg |
| Cycloheximide | 5mg |
| Chloramphenicol | 1mg |
| Bacitracine | 0.25mg |
| Agar | 15g |
| Distilled water | 1L |
June 24
We decided to make a pilot experiment in order to standardize the conditions of phcA amplification before to start with the massive screening . We choose #37 strain randomly for the standardization.
June 26
In order to standardize the conditions of phcA amplification before to start with the massive screening , we determine the annealing temperature (71°C) performing an in silico PCR (FAST PCR) and used that temperature to calculate a temperature gradient (form 64 to 71), reactions were carry out with or without DMSO. The results weren’t expected, none of the temperatures amplified.
June 27
We perfomed phcA PCRs using a boiling of the solid culture as a source of DNA. The results remained the same. Due to results we decided to change of strains and we growth on solid 3 new accessions.
