Dandekar & Chandler:Burkholderia Mating

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(Burkholderia plasmid mating protocol)
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::- LB spread with 100 ul 2M MgSO4
::- LB spread with 100 ul 2M MgSO4
::- LB + polymyxin B (pxb) at 15 ug/mL (to select for burkholderia) + plasmid selective antibiotic
::- LB + polymyxin B (pxb) at 15 ug/mL (to select for burkholderia) + plasmid selective antibiotic
-
*The day before first mating, inoculate into LB:
+
 
 +
'''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)
::1. HB101-pRK2013 (helper strain for mating)
::2. SY327-pGPI for first mating or S17.1-pDAI-SceI for second mating)
::2. SY327-pGPI for first mating or S17.1-pDAI-SceI for second mating)
::*SY327 has λ''pir'' gene required for pGPI ori R6K, but no tra genes for conjugation -- requires pRK2013 strain to help mating
::*SY327 has λ''pir'' gene required for pGPI ori R6K, but no tra genes for conjugation -- requires pRK2013 strain to help mating
::3. Burkholderia strain K56-2 (recipient)
::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'''
'''First Mating - Day 1'''
Line 79: Line 79:
::''Nicole plates 10^-3, 10^-4, and 10^-5 dilutions to prevent dealing with lawns''
::''Nicole plates 10^-3, 10^-4, and 10^-5 dilutions to prevent dealing with lawns''
:Incubate 1-3 days
:Incubate 1-3 days
-
::- Bc K56-2 colonies are small after 24 hours; Can usually work with 48 hour colonies
+
::''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
+
'''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'''
'''Second mating'''

Revision as of 16:27, 10 March 2014

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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

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

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