Difference between revisions of "Team:Newcastle/Notebook/Endophyte"
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<p><font size="3">The team planted their first set of 16 Arabidopsis seeds in 1% agar in a pipette-tip box placed on the lab windowsill. This would give an indication as to if these conditions were suitable for growth. After 7 days 12/16 seeds had germinated showing this method was appropriate.</font></p> | <p><font size="3">The team planted their first set of 16 Arabidopsis seeds in 1% agar in a pipette-tip box placed on the lab windowsill. This would give an indication as to if these conditions were suitable for growth. After 7 days 12/16 seeds had germinated showing this method was appropriate.</font></p> | ||
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<h3 class="h2">Week Beginning 30/07</h3> | <h3 class="h2">Week Beginning 30/07</h3> | ||
| − | <p><font size="3">Development of our new endophytic chassis began on the 1st of August with the arrival of root colonising Pseudomonas sp. DSM25356 from DSMZ in Germany. The strain arrived in a glass ampoule and was inoculated onto tryptone soy agar (TSA) plates using <font color=" | + | <p><font size="3">Development of our new endophytic chassis began on the 1st of August with the arrival of root colonising Pseudomonas sp. DSM25356 from DSMZ in Germany. The strain arrived in a glass ampoule and was inoculated onto tryptone soy agar (TSA) plates using <font color="blue">methods outlined by DSMZ.</font> The plates were incubated at 28 ℃ for 24 hours after which they were used to inoculate tryptone soy broth (TSB) for initial growth characterisation and antibiotic testing.</font></p> |
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<p><font size="3">In order to select transformants when introducing foreign DNA, we first needed to determine antibiotics active against Pseudomonas sp. We initially planned to transform Pseudomonas sp. with the one of the interlab test devices we modified to contain mNeonGreen instead of GFP. Creating a mutant Pseudomonas sp. with strong fluorescence would be useful for identification when carrying out microscopy on roots inoculated with Pseudomonas sp. As the test devices are in a pSB1C3 backbone, activity of chloramphenicol against Pseudomonas sp. had to be characterised. To test activity 200 µl of Pseudomonas sp. TSB overnight culture was spread onto TSA containing 50 and 100 µg/ml of chloramphenicol.</font></p> | <p><font size="3">In order to select transformants when introducing foreign DNA, we first needed to determine antibiotics active against Pseudomonas sp. We initially planned to transform Pseudomonas sp. with the one of the interlab test devices we modified to contain mNeonGreen instead of GFP. Creating a mutant Pseudomonas sp. with strong fluorescence would be useful for identification when carrying out microscopy on roots inoculated with Pseudomonas sp. As the test devices are in a pSB1C3 backbone, activity of chloramphenicol against Pseudomonas sp. had to be characterised. To test activity 200 µl of Pseudomonas sp. TSB overnight culture was spread onto TSA containing 50 and 100 µg/ml of chloramphenicol.</font></p> | ||
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<p><font size="3">As Pseudomonas sp. DSM 25356 was found to be resistant to chloramphenicol, a selection of four further antibiotics (kanamycin, gentamycin, streptomycin and carbenicillin) were test for activity against Pseudomonas sp. 20 ml of stock solution was prepared for each of the antibiotics at concentrations show in Table 1 and water or an ethanol solution was used as the solvent. Stock were distributed into 1 ml aliquots and frozen at -20 °C.</font></p> | <p><font size="3">As Pseudomonas sp. DSM 25356 was found to be resistant to chloramphenicol, a selection of four further antibiotics (kanamycin, gentamycin, streptomycin and carbenicillin) were test for activity against Pseudomonas sp. 20 ml of stock solution was prepared for each of the antibiotics at concentrations show in Table 1 and water or an ethanol solution was used as the solvent. Stock were distributed into 1 ml aliquots and frozen at -20 °C.</font></p> | ||
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Revision as of 13:12, 12 October 2018
Alternative Roots
Endophytic Chassis Notebook
NOTEBOOK
Developing Pseudomonas as a new endophytic chassis
Week Beginning 20/07
Preliminary work began with the team developing agar-based germination methods, this was mainly due to the lack of plant research experience in the team. The team conceptualised growing Arabidopsis in microcentrifuge tubes within pipette-tip boxes.
The team planted their first set of 16 Arabidopsis seeds in 1% agar in a pipette-tip box placed on the lab windowsill. This would give an indication as to if these conditions were suitable for growth. After 7 days 12/16 seeds had germinated showing this method was appropriate.
Week Beginning 30/07
Development of our new endophytic chassis began on the 1st of August with the arrival of root colonising Pseudomonas sp. DSM25356 from DSMZ in Germany. The strain arrived in a glass ampoule and was inoculated onto tryptone soy agar (TSA) plates using methods outlined by DSMZ. The plates were incubated at 28 ℃ for 24 hours after which they were used to inoculate tryptone soy broth (TSB) for initial growth characterisation and antibiotic testing.
Week Beginning 06/08
In order to select transformants when introducing foreign DNA, we first needed to determine antibiotics active against Pseudomonas sp. We initially planned to transform Pseudomonas sp. with the one of the interlab test devices we modified to contain mNeonGreen instead of GFP. Creating a mutant Pseudomonas sp. with strong fluorescence would be useful for identification when carrying out microscopy on roots inoculated with Pseudomonas sp. As the test devices are in a pSB1C3 backbone, activity of chloramphenicol against Pseudomonas sp. had to be characterised. To test activity 200 µl of Pseudomonas sp. TSB overnight culture was spread onto TSA containing 50 and 100 µg/ml of chloramphenicol.
Week Beginning 13/08
As Pseudomonas sp. DSM 25356 was found to be resistant to chloramphenicol, a selection of four further antibiotics (kanamycin, gentamycin, streptomycin and carbenicillin) were test for activity against Pseudomonas sp. 20 ml of stock solution was prepared for each of the antibiotics at concentrations show in Table 1 and water or an ethanol solution was used as the solvent. Stock were distributed into 1 ml aliquots and frozen at -20 °C.