Team:Heidelberg/Templates/Delftibactin week21


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17-09 - 22-09-13

Triple Clone pIK2.6, DelH and DelH I 6B


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:
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


File:Heidelberg Microtof0925.pdf


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.