Streptomyces:Protocols/Conjugation

 

Intergeneric Conjugation and Overlay

Description
Transfer of plasmid/cosmid DNA from a host strain, e.g. E.coli ET12567 [pUZ8002], to the recipient strain, e.g. Streptomyces coelicolor.

 

Approx. Duration:
	Conjugation preparation	~30 minutes
	Preparing the host cells	~25 minutes
	Preparing the recipient cells	~15 minutes
	Plating the conjugant mix	~20 minutes
	Whole Conjugation	~1 hour 30 minutes
	Overlay preparation	~5 minutes
	Overlaying	~10 minutes

 

Uses
Transfer a construct (cloned plasmid/cosmid) into Streptomyces to knock-out, knock-in, complement or over-express a gene.

Prerequisites
You need to know if the Streptomyces species you are using has a methyl-specific restriction mechanism. If this is the case, intergeneric conjugation into Streptomyces will require the use of the E.coli strain ET12567 [pUZ8002]; otherwise DH5α [pUZ8002] may be used. You should also know the antibiotic resistance markers that the E.coli strain, construct and Streptomyces strain confer.

Safety
General laboratory & molecular microbiology safety rules apply.

Requirements
LB/LB-NaCl, SFM+MgCl₂ , 2xYT, sterile distilled water (sdH₂ O), 15mL falcon tube(s), micro-centrifuge tube(s) (MCT), heating block @ 50°C, incubator @ 30°C, falcon centrifuge, micro-centrifuge, vortexer, Antibiotic(s).

Preparation
Pour 4 plates per conjugation (plus parallels if necessary) of SFM+MgCl₂ (10mM final) and dry for ~1 hour. Place the LB/LB-NaCl on ice. Set a heating block to 50°C. Label all tubes and plates to be used.

Method
All parts are to be performed under sterile conditions, i.e. a Bunsen burner/laminar flow hood. Spins should be done at the maximum capacity of the centrifuge/tubes. Before plating the conjugation, each MCT should be thoroughly mixed by vortexing.
Making the E.coli conjugationally competent:

Synchronising the germination of Streptomyces spores:

Initiating conjugation:

Setting up serial dilutions:

Plating:

 

To a suitable sized falcon, add:

Notes
Conjugation of a plasmid or cosmid into Streptomyces coelicolor is restricted by two main factors. Firstly, intergeneric conjugation can only be achieved if the vector contains the Streptomyces origin of replication � oriT. Secondly, the construct must be transformed into the E.coli strain ET12567 [pUZ8002]. This is due to methyl-specific restriction mechanisms that some Streptomyces use to protect themselves against the introduction of heterologous DNA. ET12567 [pUZ8002] is methylation deficient and therefore a convenient donor to use. If the Streptomyces species does not have methyl-sensing mechanisms then DH5α [pUZ8002] may be used. The plasmid pUZ8002 is required in both cases because of the tra gene, which encodes a transfer protein Tra. This enables RP4 derived in trans mobilisation of tra defective vectors in intergeneric conjugations.

The number of parallels performed depends on the type of conjugation:

 

A consideration with the E.coli strain ET12567 is its growth rate. Its doubling rate is slightly longer than DH5α. Thus the subculture needs a longer incubation (up to 4 hours) and the plates a few extra hours.

The antibiotics in the overlay select for the correct exconjugants. It is done after the incubation overnight to allow for the construct replication / integration into the genome of Streptomyces. If a construct confers Apramycin resistance then all successful exconjugants will be Apramycin resistant. When used at the suggested concentrations Nalidixic Acid bacteriostatically inhibits the growth E.coli cells. It is thought to do this by disrupting DNA repair mechanisms. This prevents E.coli from over growing the Streptomyces, allowing it to develop normally. Also, mechanical selection of a Streptomyces colony will prove easier in later steps.