Dandekar & Chandler:Burkholderia Mating

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Burkholderia Mating Protocols

Burkholderia mini-Tn7 copy site insertion protocol

From Charlotte M.

Strains needed

1. SM10 (λpir)/pTNS2

2. HB101/pRK2013

3. E. coli + mobilizable mini-Tn7 vector

4. Recipient strain

Day 1

Grow bacteria in LB with appropriate ab - Can use O/N culture or exponential growth

On pre-poured and cooled LB plates, spread 100 ul of 2M MgSO4

Prepare the following culture mixtures:

1. 100 ul each of strains 1-4

2. 100 ul recipient strain alone (control)

3. 100 ul strains 1-3 (control)

Spot 100 ul of each mixture onto LB+MgSO4 plates

Incubate overnight at 37°C

Day 2

Scrape up cells from mating plate and resuspend in 1 mL LB

Spread 200 ul aliquots onto LB plates containing the selection for mini Tn7 insert (kanamycin or zeocin) and the counter selection for your recipient strain (for burkholderia, use polymyxin B at 15ug/mL)

Incubate at 37°C for 1-5 days

Burkholderia plasmid mating protocol

Adapted from Charlotte's protocol above for use with Valvano unmarked gene deletion protocol

Assumes use of Valvano plasmids pGPI (TpR-100) and pDAI-SceI (TetR-100)

• Before starting, prepare the following plates:

- LB spread with 100 ul 2M MgSO4

- LB + polymyxin B (pxb) at 15 ug/mL (to select for burkholderia) + plasmid selective antibiotic

• The day before first mating, inoculate into LB:

1. HB101-pRK2013 (helper strain for mating)

2. SY327-pGPI for first mating or S17.1-pDAI-SceI for second mating)

• SY327 has λpir gene required for pGPI ori R6K, but no tra genes for conjugation -- requires pRK2013 strain to help mating

3. Burkholderia strain K56-2 (recipient)

First Mating - Day 0

Grow all strains in LB overnight, making sure to grow E. coli strains with the antibiotic needed to maintain plasmid

First Mating - Day 1

If exponentially growing culture is preferred, back dilute all strains with appropriate antibiotic and grow to OD ~0.4

Prepare the following culture mixtures:

1. 100 ul each of strains 1-3

2. 100 ul recipient strain alone (control)

3. 100 ul each of E. coli strains (control)

Spot 100 ul of each mixture onto LB+MgSO4 plates

Incubate overnight at 37°C

First Mating - Day 2

Scrape up cells from mating plate and resuspend in 1 mL LB

From LB suspension, make several dilutions (10^-1 to 10^-5)

Spread 200 ul aliquots onto LB plates containing pxb + Tp100

Nicole plates 10^-3, 10^-4, and 10^-5 dilutions to prevent dealing with lawns

Incubate 1-3 days

- Bc K56-2 colonies are small after 24 hours; Can usually work with 48 hour colonies

Patch single colonies onto pxb15 + Tp100 and inoculate into LB + pxb15 + Tp100

Grow overnight, then make frozen stock of potential colonies

Second mating