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Team:Heidelberg/Templates/Delftibactin week21
From 2013.igem.org
Contents |
17-09 - 22-09-13
Triple Clone pIK2.6, DelH and DelH I 6B
Electroporation
We electroporated all three plasmids again into E. coli BL21 DE3. This time, we incubated them them for 3 full days.
| Electroporation | Total amount of DNA [ng] | Ratio pIK8.6: DelH:DelRest | pIK8.6-1(12 Kb) | DelH-I6B midi (23 Kb, 235.2 ng/µl) | DelRest (32 Kb, 2.4 µg/µl) |
|---|---|---|---|---|---|
| Triple into E. coli DH10ß | 774.2 | 1:2:3 | 1 µl of 1:10 (146 ng) | 1 µl (235.2 ng) | 1.5 µl of 1:10 (375 ng) |
Preparation of Cultures for Purification and MICRO-TOF
| Bacteria | Media | Antibiotics |
|---|---|---|
| -- | ACM | -- |
| Delftia acidovorans SPH-1 | ACM | Ampicillin |
| E. coli BL21 DE3 | ACM | -- |
| E. coli BL21 DE3 with plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6 | ACM | Ampicillin, Chloramphenicol, Kanamycin |
- Inoculation of 10 ml of over night cultures of each sample
- For DH10β with plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6 no such culture was made
- Growth at 30°C
- For each culture 1 L of ACM media with antibiotics was inoculated with this pre-culture
- 1 L of ACM media with antibiotics was directly inoculated with 1 mL of a 3 hour culture DH10β with plasmids pHM04 (I6B), pFSN (D8w) & pIK8.6 which had originally been made from a clone which had been picked from the agar plate. The 1 L culture was started 3 hours later than the other cultures
- Expression was induced in E. coli BL21 DE3 with 1 mM IPTG after 8 h of growth
- Cultures were harvested after 64 hours of growth
Purification of Delftibactin
- Centrifugation of cultures at 3750 rpm for 30 min
- Retrieved supernatant
- Added 20 g/L HP-20
- Stirred at RT for 2 h
- Filtration with Buchner funnel to retain HP-20
- Washed with 400 ml dddH2O
- Elution with 400 ml Methanol
- Evaporated methanol with rotary evaporator
- Resuspended in 2 ml 50 methanol : 50 ddH2O
MICRO-TOF
File:Heidelberg Microtof0925.pdf
Result
Only in the culture of D. acidovorans SPH-1 Delftibactin could be detected. The samples were negative for delftibactin. Apparently our triple clone does not produce delftibactin. However, this is not surprising, since the DelH clone I 6B harbors a mutation in the promoter region.
