Dandekar & Chandler:Burkholderia Mating: Difference between revisions
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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 | ||
'''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 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'' | ||
'''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 14:27, 10 March 2014
Back to Protocols
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