Difference: ProtocolsWorkingWithSerratiaSymbiotica (5 vs. 6)

Revision 62022-08-10 - KateElston

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+ Working with _Serratia symbiotica
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+ Working with Serratia symbiotica
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 Serratia symbiotica is a secondary endosymbiont of the black bean aphid ( Aphis fabae) gut microbiota that can be cultivated in vitro. The isolation and characterization of the particular strain used were carried out by Dr. Ahmed Sabri at the University of Liege, Belgium and are described in depth in here.

 In vitro culture conditions
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+ Plate Growth
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+ Serratia symbiotica DSMZ Strain: 23270 (type strain) can be cultured anywhere from 25-30°C ; although recommended culturing temperature found in literature is 28°C. It grows robustly on agar plates after 2-3 days on the following media:
  
 Tripticase Soy Agar
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+Plate Growth 

 Serratia symbiotica DSMZ Strain: 23270 (type strain) can be cultured anywhere from 25-30°C. I typically grow it at room temperature, although recommended culturing temperature found in literature is 28°C. It grows robustly on agar plates after 2-3 days on the following media: 
 Tripticase Soy Agar (see below for prep)
 



Liquid Media 

Serratia symbiotica can be cultivated in the following liquid media (same general culture conditions as above, with shaking): 

 Tripticase Soy Broth (see below for prep)
 

*Doubling time in media at 25°C is ~4 hours
 Antibiotics: 
 Resistant to Vancomycin
  Gentamycin should be used at a concentration of 40 μg/mL
  Sensitive to all other working antibiotic concentrations used for E. coli
 

Heat Sensitivity:
 

 Don't leave plates to dry near flame for extended periods of time; S. symbiotica is heat sensitive and will lyse quickly
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+ Liquid Media
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+ Media Prep
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+Commercial Trypticase soy broth powder has sugars in it that will burned if autoclaved. Therefore it is important to use the method below to prepare it.
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+Serratia symbiotica can be cultivated in the following liquid media (same general culture conditions as above, with shaking): 
 Tripticase Soy Broth
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+TSB (0.5 L) 
 15 g TSB Powder
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+.5 L Distilled Water
 
- filter sterilize components into sterile/autoclaved bottle
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+*Doubling time in media at 25°C is ~4 hours
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+TSA (1 L)
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+g Agar
  333 mL Distilled Water
 
- autoclave the agar mixture
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+ Transforming _S. symbiotica
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+g TSB Powder
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+mL Distilled Water
 
- filter sterilize and mix with autoclaved water/agar (I tend to filter sterilize straight into the water agar bottle, if possible)
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+ Transforming S. symbiotica
  Electroporation protocol 

Making S. symbiotica electrocompetent: (protocol based on E. coli version: http://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells) 

 Grow up S. symbiotica for two days from glycerol stock at room temperature (shaking)
  Innoculate 50ul culture into 50ml TSB in 250ml flask
  Grow until OD ~ 0.4 (check after 16 hours)
  Transfer 25 ml into two Falcon tubes
  Centrifuge at 6000rpm for 5min at 4°C
  Wash with 20 ml 10% glycerol
  Repeat wash step 4 more times
  Resuspend in 250ul 10% glycerol
  Divide into 50ul aliquots and freeze at -80 or continue electroporation protocol
 

Electroporation of S. symbiotica : 

 Allow -80 aliquots to thaw on ice
  Add 2ul plasmid to 50ul cells, transfer to ice cold electroporation cuvette
  Electroporate using Ec2 setting (2.5V)
  Quickly add in 950ul TSB and allow to recover for at least 4 hours (overnight is even better)
  Spin down at 3000rpm for 5min
  Resuspend pellet in 50ul TSB and plate all on desired antibiotic plate
  Allow to grow at room temp (colonies can appear as early as 2 days)
 
 Electroporation Notes
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+ Both of these protocols will be updating as I improve them!
  When preparing electrocompetent cells all steps should be performed on ice
  In preparing electrocompetent cells, ODs very close to 0.4 work well, other ODs have yet to be tested but range between 0.4-0.6 should work
  For the electroporation itself colonies can take up to 7 days to appear; as I update the protocol this should improve, but not to worry if your cells are slow to grow!
 

-- KateElston - 28 Apr 2017
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