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 for Bc/TpR-20 for E. coli) and pDAI-SceI (TetR-100/TetR-20); also uses pRK2013 (KanR-35)

  • 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

First Mating - Day 0

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

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 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

First Mating - Day 3

Patch single colonies onto pxb15 + Tp100 and inoculate into LB + pxb15 + Tp100
Grow overnight, then make frozen stock of potential colonies



Second Mating - Day 1

Inoculate into LB + appropriate antibiotic and grow overnight:
Bc construct from previous mating (Tp100)
HB101-pRK2013 (Kan35)
S17-1 pDAI-SceI (Tet20)

Second Mating - Day 2

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

Second Mating - Day 3

Scrape up cells from mating plate and resuspend in 1 mL LB
From LB suspension, make 10x and 100x dilutions
Spread 200 ul aliquots onto LB plates containing pxb + Tp100
Nicole experience is very low efficiency for this second mating; suggest doing 200 ul pellet suspension, 200 ul each of the dilutions - will probably only get positive colonies on the pellet plate
Incubate 1-3 days
Bc K56-2 colonies are small after 24 hours; Can usually work with 48 hour colonies

Second Mating - Day 4

Patch single colonies onto pxb15 + Tet100
Incubate overnight at 37°C

Second Mating - Day 5

Patch single colonies again, onto LB + Tet100
Incubate overnight at 37°C

Second Mating - Day 6

Patch positive colonies onto LB + Tet100, LB +Tp100 and LB alone
Incubate overnight at 37°C
  • Want to verify integration of the first plasmid by ensuring the loss of Tp resistance; positive colonies will continue to grow on Tet100 but will not grow on Tp100

Second Mating - Day 7

Select positive colonies that do not grow on Tp to screen for the correct deletion
Make frozen stock of positive colonies
Passage positive colonies from the LB plate to promote second plasmid loss
Not sure yet if this is better to do on plates or in liquid culture; so far 3 passages on LB plates is not enough to force the loss of pDAI, evidenced by continuation of growth on Tet100 (Deletion in gene of interest IS present, just can't get the strain to kick out pDAI!)
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