Working with Serratia symbiotica

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. Engineering methods for this strain are described in Elston et al., 2021.

In vitro culture conditions

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.

Media Prep

Commercial Trypticase soy broth powder has sugars in it that will burned if autoclaved. Therefore, it is important to sterilize it with vacuum filtration.

TSB (0.5 L)

• 15 g TSB Powder
• 0.5 L Distilled Water

- filter sterilize components into sterile/autoclaved bottle

TSA (0.5 L)

• 8 g Agar
• 165 mL Distilled Water

- autoclave the agar mixture

• 16 g TSB Powder
• 335 mL Distilled Water

- filter sterilize and mix with autoclaved water/agar (I tend to filter sterilize straight into the water agar bottle, if possible)

Transforming S. symbiotica

S. symbiotica can be transformed with conjugation and electroporation. Conjugation is described here and the electroporation protocol is below.

Electroporation protocol

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

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

Electroporation of S. symbiotica :

1. Allow -80 aliquots to thaw on ice
2. Add 2ul plasmid to 50ul cells, transfer to ice cold electroporation cuvette
3. Electroporate using Ec2 setting (2.5V)
4. Quickly add in 950ul TSB and allow to recover for at least 4 hours (overnight is even better)
5. Spin down at 3000rpm for 5min
6. Resuspend pellet in 50ul TSB and plate all on desired antibiotic plate
7. Allow to grow at room temp (colonies can appear as early as 2 days)

Electroporation Notes

• 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!

-- Kate Elston - 2022-08-11