(r8) Barrick Lab :: ProtocolsWorkingWithSerratiaSymbiotica

---+ Working with <i>Serratia symbiotica</i>


_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 [[http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.024133-0#tab2][here]]. Engineering methods for this strain are described in [[https://journals.asm.org/doi/10.1128/aem.02245-20?permanently=true][Elston et al., 2021]].

---++ _In vitro_ culture conditions
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*Plate Growth* </br> 
 _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* </br>
_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 &#956;g/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; *<i>S. symbiotica</i> 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.

*<u>TSB (0.5 L)</u>*
   * 15 g TSB Powder
   * 0.5 L Distilled Water
- filter sterilize components into sterile/autoclaved bottle

*<u>TSA (0.5 L)</u>*
   * 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 <i>S. symbiotica</i>
_S. symbiotica_ can be transformed with conjugation and electroporation. Conjugation is described [[ProtocolsConjugation][here]] and the electroporation protocol is below.

---+++++ Electroporation protocol

<strong>Making _S. symbiotica_ electrocompetent:</strong> (protocol based on <em>E. coli </em>version: [[ProtocolsElectrocompetentCells][http://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells]]<em>)</em>
   1 Grow up _S. symbiotica_ for two days from glycerol stock at room temperature (shaking)</span>
   1 Innoculate 50ul culture into 50ml TSB in 250ml flask
   1 Grow until OD ~ 0.4 (check after 16 hours)
   1 Transfer 25 ml into two Falcon tubes
   1 Centrifuge at 6000rpm for 5min at 4°C
   1 Wash with 20 ml 10% glycerol
   1 Repeat wash step 4 more times
   1 Resuspend in 250ul 10% glycerol
   1 Divide into 50ul aliquots and freeze at -80 or continue electroporation protocol

*Electroporation of _S. symbiotica_ :*
   1 Allow -80 aliquots to thaw on ice
   1 Add 2ul plasmid to 50ul cells, transfer to ice cold electroporation cuvette
   1 Electroporate using Ec2 setting (2.5V)
   1 Quickly add in 950ul TSB and allow to recover for at least 4 hours (overnight is even better)
   1 Spin down at 3000rpm for 5min
   1 Resuspend pellet in 50ul TSB and plate all on desired antibiotic plate
   1 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!

-- %USERSIG{KateElston - 2022-08-11}%
Barrick Lab > ProtocolList > ProtocolsWorkingWithSerratiaSymbiotica
Contributors to this topic
KateElston, AnthonyVandieren
Topic revision: r8 - 2022-08-11 - 17:54:49 - Main.KateElston