MFeeney: /* Notes */

2022-03-20T23:34:01Z

Notes

← Older revision		Revision as of 23:34, 20 March 2022
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	===Notes===		===Notes===
	# You should confirm the genotype of your transductants using an appropriate method (PCR, Southern blotting, whole genome sequencing.)		# You should confirm the genotype of your transductants using an appropriate method (e.g. PCR, Southern blotting, whole genome sequencing.)



	===References===		===References===

MFeeney at 23:32, 20 March 2022

2022-03-20T23:32:31Z

← Older revision		Revision as of 23:32, 20 March 2022
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	===Phage transduction===		===Phage transduction===

			SV1 is the best-established transducing phage for use in <em>Streptomyces</em> and is specific for use with <em>S. venezuelae</em>.<sup>[1]</sup>

			Phage transduction is useful for moving alleles (usually marked with an antibiotic resistance marker or other selectable marker) into another genetic background. For example, after deleting a gene using PCR targeting (a.k.a. "Redirect") it is often best to move this allele into a clean wild-type background (for an example, see [2]).


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	Materials needed:		Materials needed:
	#		# Soft nutrient agar ([[SNA]])
			# [[DNA]] agar plates
			# [[DNB]]
			# [[MYM]] or another appropriate medium for growth of your strains
			# Antibiotic needed to select for the allele being transduced
			# SV1 phage (see [[High titre preparation of phage]] for how to prepare this)
			# Spore stocks of the appropriate <em>S. venezuelae</em> strains (e.g., the donor stain containing the allele you wish to transduce, and the recipient strain that you wish to move this allele into)
			# Sterile glucose (i.e. 20% w/v glucose)
			# Sterile MgSO<sub>4</sub> stock solution
			# Sterile Ca(NO<sub>3</sub>)<sub>2</sub> stock solution
			# 0.45 μm filters
			# Spreaders, pipettes, toothpicks, Eppendorf tubes, etc.

	Instructions:		Instructions:
	#		# You should titer your spore stock and phage before beginning.
			# First, prepare a lysate of the SV1 phage on your donor strain (the strain containing the allele you wish to transduce):
			## Prepare [[DNA]] plates containing 0.5% glucose, 10 mM MgSO<sub>4</sub> and 10 mM Ca(NO<sub>3</sub>)<sub>2</sub> and molten [[SNA]] (not too hot - should be ~45°C)
			## Add 10<sup>4</sup> of SV1 phage to 10<sup>6</sup> donor spores in 800 μl pre-warmed (45°C) soft nutrient agar (SNA)
			## Pour this mixture onto your DNA/glucose/MgSO<sub>4</sub>/Ca(NO<sub>3</sub>)<sub>2</sub> plates and let the molten agar solidify.
			## Incubate the plates at 30°C overnight.
			## Harvest the phage by flooding the plate with 2.5 ml DNB and incubate for 3-4 hr at room temperature.
			## Collect the phage-containing DNB soak-out and filter through a 0.45 μm filter to eliminate bacterial contamination.

			# To then transduce your marked allele into the recipient strain:
			## First, titer the new phage lysate and the spore stock of the recipient strain
			## Mix 10<sup>9</sup> phage with 10<sup>7</sup>-10<sup>8</sup> spores of the recipient strain
			### Controls here should be phage alone, and recipient strain alone.
			## Spread on MYM agar (or another appropriate medium depending on your strain's growth requirements).
			## Incubate overnight at room temperature before overlaying with the appropriate antibiotic that will select for the mutant allele you are transducing.
			## Incubate plates at 30°C until transductants appear. These should be restreaked several times on appropriate medium (e.g. MYM) to confirm their phenotype (antibiotic resistance) and to purify away any remaining phage particles.

	===Notes===		===Notes===
	#		# You should confirm the genotype of your transductants using an appropriate method (PCR, Southern blotting, whole genome sequencing.)


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	===References===		===References===

			[1] Stuttard, C. “Transduction of auxotrophic markers in a chloramphenicol-producing strain of Streptomyces.” Journal of general microbiology vol. 110,2 (1979): 479-82. doi:10.1099/00221287-110-2-479

	~~Protocol adapted for ActinoBase by Morgan Feeney from the University of Strathclyde~~.		[2] Tschowri, Natalia et al. “Tetrameric c-di-GMP mediates effective transcription factor dimerization to control Streptomyces development.” Cell vol. 158,5 (2014): 1136-1147. doi:10.1016/j.cell.2014.07.022



			Protocol adapted for ActinoBase by Morgan Feeney from the University of Strathclyde.


	To prepare SV1-lysate, 104 phage were added to 106 SV77 donor spores in 800 μl pre-warmed (45°C) soft nutrient agar (SNA) and poured onto Difco nutrient agar plates containing 0.5% glucose, 10 mM MgSO4 and 10 mM Ca(NO3)2. The plates were incubated at 30°C overnight, then flooded with 2.5 ml Difco nutrient broth (DNB) and incubated for 3-4 hr at room temperature. The phage-containing DNB soak-out was harvested and filtered through a 0.45 μm filter to eliminate bacterial contamination. For transduction of the bldD::apr allele, 109 phage particles harvested from the bldD::apr mutant strain SV77, were mixed with 107-108 WT spores and incubated overnight on MYM agar at room temperature before overlaying with apramycin for selection. Plates spread with the recipient strain or the phage alone were used as controls. Transduction of ~~the bldD::apr allele was confirmed by PCR using test primers listed in Table S4, and the strain was named SV74~~.

MFeeney: Created page with "=Phage transduction using SV1= This protocol has been confirmed to work for the following organisms: **Streptomyces venezuelae* with ΦSV1 ===Ph..."

2022-03-20T21:50:08Z

Created page with "=Phage transduction using SV1= This protocol has been confirmed to work for the following organisms: *Streptomyces venezuelae with ΦSV1 ===Ph..."

New page

=Phage transduction using SV1=

This protocol has been confirmed to work for the following organisms:
*[[Streptomyces venezuelae]] with ΦSV1

===Phage transduction===

===Protocol===

Materials needed:
#

Instructions:
#

===Notes===
#

===References===

Protocol adapted for ActinoBase by Morgan Feeney from the University of Strathclyde.

To prepare SV1-lysate, 104 phage were added to 106 SV77 donor spores in 800 μl pre-warmed (45°C) soft nutrient agar (SNA) and poured onto Difco nutrient agar plates containing 0.5% glucose, 10 mM MgSO4 and 10 mM Ca(NO3)2. The plates were incubated at 30°C overnight, then flooded with 2.5 ml Difco nutrient broth (DNB) and incubated for 3-4 hr at room temperature. The phage-containing DNB soak-out was harvested and filtered through a 0.45 μm filter to eliminate bacterial contamination. For transduction of the bldD::apr allele, 109 phage particles harvested from the bldD::apr mutant strain SV77, were mixed with 107-108 WT spores and incubated overnight on MYM agar at room temperature before overlaying with apramycin for selection. Plates spread with the recipient strain or the phage alone were used as controls. Transduction of the bldD::apr allele was confirmed by PCR using test primers listed in Table S4, and the strain was named SV74.

SV1 transduction - Revision history

MFeeney: /* Notes */

MFeeney at 23:32, 20 March 2022

MFeeney: Created page with "=Phage transduction using SV1= This protocol has been confirmed to work for the following organisms: *Streptomyces venezuelae with ΦSV1 ===Ph..."

MFeeney: Created page with "=Phage transduction using SV1= This protocol has been confirmed to work for the following organisms: **Streptomyces venezuelae* with ΦSV1 ===Ph..."