Team:Heidelberg/Templates/Delftibactin week23

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Contents

30-09 - 06-10-13

Triple Clone pIK.6, DelRest and DelH C5

Electroporation

E. coli BL21 DE3 + DelRest + pIK8.6 were electroporated with DelH C5 miniprep DNA and streaked on 2YT + Amp + Chlor + Kan plates and incubated at RT for 2 days.

Evaluation of Gold Precipitation on ACM Plates

In order to evaluate gold nanoparticle formation capacity DelH clone C5 in presence of DelRest and pIK8.6, they were streaked on ACM plates as follows and incubated at 30°C for 3 days. As controls, we used the BL21 DE3 transformed with pSB3C5 and pSB6A1.

Plate Antibiotic Cells
ACM - D.acidovorans
E.coli BL21 DE3 + pSB3C5 + pSB6A1
E.coli BL21 DE3 + DelH C5 + DelRest + pIK8.6
empty
ACM Amp + Chlor + Kan D.acidovorans
E.coli BL21 DE3 + pSB3C5 + pSB6A1
E.coli BL21 DE3 + DelH C5 + DelRest + pIK8.6
empty
ACM + IPTG Amp + Chlor + Kan D.acidovorans
E.coli BL21 DE3 + pSB3C5 + pSB6A1
E.coli BL21 DE3 + DelH C5 + DelRest + pIK8.6
empty

Following 3 day incubation, 200 µl 5 mM gold chloride in soft agar was applied.

Result

Evaluation of Gold Precipitation of C5 Triple Clone on ACM Plates following 3 day Incubation at 30°C

Fig.1.1 From left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans prior to addition of gold chloride soft agar.

Fig.1.2 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 0:00.

Fig.1.3 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 0:15.

Fig.1.4 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 0:30.

Fig.1.5 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 1:00.

Fig.1.6 rom left: background control, E.coli BL21 DE3 containing ampicillin and chloramphenicol parts, E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 and D.acidovorans: T= 1:30.

Unexpectedly, the D.acidovorans control grew on the ACM + Amp + Chlor + Kan plates, indicating a contamination with triple resistant bacteria. Therefore, the experiment is inconclusive and has to be repeated. Moreover, the background control shows significant activity.
Nevertheless, there was neither increased activity of the induced 'E.coli BL21 DE3 containing DelH C5, DelRest and pIK8.6 compared to the E.coli BL21 DE3 negative control nor to the not induced control.

Preparation of Cultures for Purification and MICRO-TOF

Bacteria Media Antibiotics
E. coli BL21 DE3 ACM --
Delftia acidovorans SPH-1 ACM --
E. coli BL21 DE3 with plasmids pHM04 (C5), pFSN (D8w) & pIK8.6 ACM Ampicillin, Chloramphenicol, Kanamycin
  • Inoculation of 10 ml of over night cultures of each sample
  • Growth at 30°C
  • For each culture, 1 L of ACM media with antibiotics was inoculated with the pre-culture
  • Expression was induced in all E. coli BL21 DE3 with 1 mM IPTG after 24 h of growth
  • All cultures were supplemented with 10 mM sodium propionate after 24 h of growth
  • The cultures selected with ampicillin were supplemented with an additional 1:2000 ampicillin after 24 h of growth
  • Cultures were harvested after 48 hours of growth and 24 hours after induction

Purification of Delftibactin

  • Centrifugation of cultures at 3750 rpm for 45 min

Protocol for liquid culture

  • 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

Protocol for pellet

  • Kept pellet
  • Washed pellet with destilled water
  • Resuspended pellet in 50 ml destilled water
  • Freeze and thawed 8 times
  • Centrifugation at 3750 rpm for 30 min
  • Kept 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 20131004 MicroTOF.pdf

Result

Unexpectedly, the D. acidovorans positive control was negative for expression of Delftibactin. The results are therefore inconclusive and the experiment has to be repeated.

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