Lambda-red mediated recombination using ssDNA - Revision history

MFeeney: /* References */

2020-05-07T14:38:05Z

References

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	[4]Feeney, M.A., Chandra, G., Findlay, K.C., Paget, M.S.B., & Buttner, M.J. (2017). Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon. mBio, 8(3) e00815-17; DOI: 10.1128/mBio.00815-17		[4]Feeney, M.A., Chandra, G., Findlay, K.C., Paget, M.S.B., & Buttner, M.J. (2017). Translational Control of the SigR-Directed Oxidative Stress Response in Streptomyces via IF3-Mediated Repression of a Noncanonical GTC Start Codon. mBio, 8(3) e00815-17; DOI: 10.1128/mBio.00815-17




	Protocol developed & written by Dr. Morgan Feeney, John Innes Centre, based on the [[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]] and the Court lab protocols for ssDNA recombineering.		Protocol developed & written by Dr. Morgan Feeney, John Innes Centre, based on the [[Lambda-red mediated recombination (PCR-targeting system a.k.a. "Redirect")]] and the Court lab protocols for ssDNA recombineering.

MFeeney: /* Step 5. Screen for successful recombinants */

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Step 5. Screen for successful recombinants

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	Instructions:		Instructions:
	*The details of your screen will vary depending on the design in Step 1 above.		*The details of your screen will vary depending on the design in Step 1 above. (See note 1.)
	*If using oligo100, pick transformants that are dark red/have dark red sectors, to screen for your mutation(s). I usually find that screening ~7-15 transformants is sufficient.		*If using oligo100, pick transformants that are dark red/have dark red sectors, to screen for your mutation(s). I usually find that screening ~7-15 transformants is sufficient.
	*Use the control plated above (untransformed HME68/cosmid strain) as a control for your diagnostic restriction digest/mismatch PCR; this strain carries the wild-type allele of your gene.		*Use the control plated above (untransformed HME68/cosmid strain) as a control for your diagnostic restriction digest/mismatch PCR; this strain carries the wild-type allele of your gene.
	*If using a diagnostic restriction digest, note that some enzymes are active in PCR buffer - see for example, [https://international.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers Activity of Restriction Enzymes in PCR buffers]
	*Select 2-4 positive candidates to proceed with step 6.		*Select 2-4 positive candidates to proceed with step 6.

			Notes:
			#If using a diagnostic restriction digest, note that some enzymes are active in PCR buffer - see for example, [https://international.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers Activity of Restriction Enzymes in PCR buffers]
			#If you are having any difficulties with your PCR screen, refer to the [[High GC PCR]] tips page.

	====Step 6. Transform DH5a with your recombineered cosmid/plasmid====		====Step 6. Transform DH5a with your recombineered cosmid/plasmid====

MFeeney: /* Step 9. Conjugation into Streptomyces */

2020-05-07T14:34:35Z

Step 9. Conjugation into Streptomyces

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	Materials needed:		Materials needed:
	*ET12567/pUZ8002,cosmid strain(s) from step 8		*ET12567/pUZ8002,cosmid strain(s) from step 8
	*~~Antibiotics~~ (here, apramycin and nalidixic acid) and reagents as detailed in the [[Conjugation using ET12567/pUZ8002]] protocol		*[[Antibiotic stocks and working concentrations\|Antibiotic solutions]] (here, apramycin and nalidixic acid) and reagents as detailed in the [[Conjugation using ET12567/pUZ8002]] protocol
	*[[MS]] solid medium or another medium suitable for growth of your ''Streptomyces'' strain		*[[MS]] solid medium or another medium suitable for growth of your ''Streptomyces'' strain

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	#Pick '''single colonies''' and restreak once on medium containing apra and nal.		#Pick '''single colonies''' and restreak once on medium containing apra and nal.
	#Restreak ex-conjugants several times on non-selective medium (i.e. on [[MS]] for ''S. coelicolor''). This will allow the wild-type/mutant alleles to assort into single copy such that you end up with exconjugants that carry only either the wild-type <strong>or</strong> the mutant allele. (Depending on the selective pressure for the wild-type/mutant alleles, you may need to restreak several times to see the wild-type allele completely eliminated from the exconjugants.)		#Restreak ex-conjugants several times on non-selective medium (i.e. on [[MS]] for ''S. coelicolor''). This will allow the wild-type/mutant alleles to assort into single copy such that you end up with exconjugants that carry only either the wild-type <strong>or</strong> the mutant allele. (Depending on the selective pressure for the wild-type/mutant alleles, you may need to restreak several times to see the wild-type allele completely eliminated from the exconjugants.)
	#Screen for apra<sup>S</sup> exconjugants (those in which a double-crossover has occurred.)		#Screen for apra<sup>S</sup> exconjugants (those in which a double-crossover has occurred.)

	====Step 10. Identify mutant exconjugants.====		====Step 10. Identify mutant exconjugants.====

MFeeney: /* Step 8. Transform ET12567/pUZ8002 with the mutant cosmid/plasmid. */

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Step 8. Transform ET12567/pUZ8002 with the mutant cosmid/plasmid.

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	*The cosmids carrying the mutant allele(s) (from step 7 above)		*The cosmids carrying the mutant allele(s) (from step 7 above)
	*Solid and liquid [[LB]] medium - see note 2		*Solid and liquid [[LB]] medium - see note 2
	*Antibiotic solutions (here, apramycin and carbenicillin)		*[[Antibiotic stocks and working concentrations\|Antibiotic solutions]] (here, apramycin and carbenicillin)
	*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation		*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation

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	#Transform ET12567/pUZ8002 with your cosmids using electroporation. Plate on LB/apra or LB/carb.		#Transform ET12567/pUZ8002 with your cosmids using electroporation. Plate on LB/apra or LB/carb.
	#Restreak transformants (on LB/apra or LB/carb)		#Restreak transformants (on LB/apra or LB/carb)


	====Step 9. Conjugation into ''Streptomyces''====		====Step 9. Conjugation into ''Streptomyces''====

MFeeney: /* Step 6. Transform DH5a with your recombineered cosmid/plasmid */

2020-05-07T14:33:44Z

Step 6. Transform DH5a with your recombineered cosmid/plasmid

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	*Cosmid candidates selected above		*Cosmid candidates selected above
	*Solid and liquid [[LB]] medium		*Solid and liquid [[LB]] medium
	*Antibiotic solutions (here, apramycin and carbenicillin)		*[[Antibiotic stocks and working concentrations\|Antibiotic solutions]] (here, apramycin and carbenicillin)
	*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation		*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation
	*Miniprep kit or reagents/supplies needed for [[cosmid DNA purification]]		*Miniprep kit or reagents/supplies needed for [[cosmid DNA purification]]

MFeeney: /* Step 5. Screen for successful recombinants */

2020-05-07T14:33:09Z

Step 5. Screen for successful recombinants

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	*The oligos designed in Step 1 above (for diagnostic restriction digest or mismatch PCR)		*The oligos designed in Step 1 above (for diagnostic restriction digest or mismatch PCR)
	*Solid and liquid [[LB]] medium		*Solid and liquid [[LB]] medium
	*Antibiotic solutions (here, apramycin and carbenicillin)		*[[Antibiotic stocks and working concentrations\|Antibiotic solutions]] (here, apramycin and carbenicillin)
	*PCR reagents and kit, restriction enzymes, etc. as needed		*PCR reagents and kit, restriction enzymes, etc. as needed

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	*If using a diagnostic restriction digest, note that some enzymes are active in PCR buffer - see for example, [https://international.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers Activity of Restriction Enzymes in PCR buffers]		*If using a diagnostic restriction digest, note that some enzymes are active in PCR buffer - see for example, [https://international.neb.com/tools-and-resources/usage-guidelines/activity-of-restriction-enzymes-in-pcr-buffers Activity of Restriction Enzymes in PCR buffers]
	*Select 2-4 positive candidates to proceed with step 6.		*Select 2-4 positive candidates to proceed with step 6.


	====Step 6. Transform DH5a with your recombineered cosmid/plasmid====		====Step 6. Transform DH5a with your recombineered cosmid/plasmid====

MFeeney: /* Step 4. ssDNA recombineering */

2020-05-07T14:32:42Z

Step 4. ssDNA recombineering

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	*Solid and liquid [[LB]] medium - see note 2		*Solid and liquid [[LB]] medium - see note 2
	*MacConkey/1% galactose medium (if using oligo100)		*MacConkey/1% galactose medium (if using oligo100)
	*Antibiotic solutions (here, apramycin and carbenicillin)		*[[Antibiotic stocks and working concentrations\|Antibiotic solutions]] (here, apramycin and carbenicillin)
	*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation		*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation

MFeeney: /* Step 3. Transform the recombineering strain with the cosmid/plasmid */

2020-05-07T14:32:15Z

Step 3. Transform the recombineering strain with the cosmid/plasmid

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	*The cosmid engineered to contain ''oriT'' in Step 2 above		*The cosmid engineered to contain ''oriT'' in Step 2 above
	*Solid and liquid [[LB]] medium		*Solid and liquid [[LB]] medium
	*Antibiotic solutions (here, apramycin and carbenicillin)		*[[Antibiotic stocks and working concentrations\|Antibiotic solutions]] (here, apramycin and carbenicillin)
	*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation		*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation

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	#Plate the transformation mixture on LB/apra/carb (30°C).		#Plate the transformation mixture on LB/apra/carb (30°C).
	#Restreak transformant(s) on LB/apra/carb (30°C). You have now generated the strain needed to perform the ssDNA recombineering.		#Restreak transformant(s) on LB/apra/carb (30°C). You have now generated the strain needed to perform the ssDNA recombineering.



	====Step 4. ssDNA recombineering====		====Step 4. ssDNA recombineering====

MFeeney at 14:31, 7 May 2020

2020-05-07T14:31:32Z

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	*Cosmid selected above		*Cosmid selected above
	*Solid and liquid [[LB]] medium		*Solid and liquid [[LB]] medium
	*Antibiotic solutions (here, kanamycin, carbenicillin, and apramycin)		*[[Antibiotic stocks and working concentrations\|Antibiotic solutions]] (here, kanamycin, carbenicillin, and apramycin)
	*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation		*Electroporation cuvettes, [[SOC]], other supplies needed for electroporation
	*Miniprep kit or reagents/supplies needed for [[cosmid DNA purification]]		*Miniprep kit or reagents/supplies needed for [[cosmid DNA purification]]

MFeeney: /* Detailed Protocols */

2020-05-07T14:29:59Z

Detailed Protocols

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 Materials needed:
 Instructions:
 ==References==