Difference between revisions of "Conjugation using ET12567/pUZ8002"

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(Intructions)
 
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#Dilute the overnight culture 1:100 in fresh [[LB]] + antibiotic selection and grow to an OD<sub>600</sub> of 0.4-0.6.
 
#Dilute the overnight culture 1:100 in fresh [[LB]] + antibiotic selection and grow to an OD<sub>600</sub> of 0.4-0.6.
 
#Wash the cells twice with an equal volume of [[LB]] (to remove antibiotics) and resuspend in 0.1 volume of [[LB]]
 
#Wash the cells twice with an equal volume of [[LB]] (to remove antibiotics) and resuspend in 0.1 volume of [[LB]]
#While washing the ''E. coli'' cells, for each conjugation add approximately 10<sup>8</sup> ''Streptomyces'' spores to 500 µl [[2X YT]] broth, heat shock at 50° for 10 minutes then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
+
#While washing the ''E. coli'' cells, for each conjugation add approximately 10<sup>8</sup> ''Streptomyces'' spores to 500 µl [[2X YT]] broth and germinate via heat shock at 50° for 10 minutes, then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and thn resuspend the pellet in the residual liquid.
+
#Add 500 µl of ''E. coli'' cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and then resuspend the pellet in the residual liquid.
 
#Plate out on [[SFM | MS]] agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9. (Note: for <em>[[Streptomyces venezuelae]]</em> incubate for 16-20 h at room temperature to avoid overgrowth which may make antibiotic selectiom more challenging)
 
#Plate out on [[SFM | MS]] agar + 10 mM MgCl<sub>2</sub> and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9. (Note: for <em>[[Streptomyces venezuelae]]</em> incubate for 16-20 h at room temperature to avoid overgrowth which may make antibiotic selectiom more challenging)
 
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
 
#Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
 
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.
 
#Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.

Latest revision as of 23:17, 25 March 2020

Intergeneric transfer of plasmids from E. coli to Streptomyces strains was demonstrated after modifying the mobilisation system established by Simon et al. (1983) for Gram-negative bacteria. It works well also for non-replicative vectors and integrate into the chromosome either site-specifically at the φC31 or pSAM2 attachment sites, or via insert-directed homologous recombination. Vectors containing oriT require transfer functions to be supplied by the E. coli donor strain such as the methylation-deficient ET12567 (MacNeil et al., 1991) carrying the non-transmissible pUZ8002.

Materials

Strains

  • E. coli donor strain ET12567/pUZ8002 containing vector
  • Acceptor Streptomyces strain

Instructions

  1. Transform ET12467/pUZ8002 with oriT-containing vector, and select for the incoming plasmid only
  2. Inoculate a colony in to 10 ml LB containing chloramphenicol, kanamycin and the antibiotic used to select for the oriT containing plasmid and grow overnight. Chloramphenicol and kanamycin sensitive segregants arise frequently among transformants, and therefore it is recommended that more than one culture is set up.
  3. Dilute the overnight culture 1:100 in fresh LB + antibiotic selection and grow to an OD600 of 0.4-0.6.
  4. Wash the cells twice with an equal volume of LB (to remove antibiotics) and resuspend in 0.1 volume of LB
  5. While washing the E. coli cells, for each conjugation add approximately 108 Streptomyces spores to 500 µl 2X YT broth and germinate via heat shock at 50° for 10 minutes, then allow to cool. If using mycelial fragments for conjugation, harvest from a 3-4 day old culture using 2-3 ml of 20% glycerol. Vortex thoroughly and use 0.5 ml of the mycelial fragments for each conjugation, omitting the heat shock. This can be varied based on conjugation efficiency.
  6. Add 500 µl of E. coli cells 500 µl heat-shocked spores or mycelial fragments. Mix and spin briefly. Pour off most of the supernatant and then resuspend the pellet in the residual liquid.
  7. Plate out on MS agar + 10 mM MgCl2 and incubate at 30° for 16-20 h. Drying the MS for 1 hour in a laminar flow hoof before plating can assist absorption of the solution used in step 9. (Note: for Streptomyces venezuelae incubate for 16-20 h at room temperature to avoid overgrowth which may make antibiotic selectiom more challenging)
  8. Overlay the plate with 1 ml water containing 0.5 mg nalidixic acid and the plasmid selection antibiotic (e.g 1 mg Apramycin) using a spreader to distribute the antibiotic solution evenly. Continue incubation at 30°
  9. Pick of potential exconjugants to media containing nalidixic acid and antibiotic selection.