(r2) ProtocolsWorkingWithSerratiaSymbiotica < Lab

---+ 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 [[http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.024133-0#tab2][here]].

---++ _In vitro_ culture conditions
---+++++ Plate Growth
 _Serratia symbiotica_ DSMZ Strain: 23270 (type strain) can be cultured 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:<br /> 
   * Tripticase Soy Agar

*Antibiotics:*
   * Resistant to Vancomycin
   * Sensitive to all other working antibiotic concentrations used for _E. coli_
---+++++ Liquid Media

_Serratia symbiotica_ can be cultivated in the following liquid media (same general culture conditions as above, with shaking):
   * Tripticase Soy Broth

*Doubling time in media is ~4 hours

---++ Transforming _S. symbiotica_

---+++++ Electroporation protocol

<strong>Making <em>S. symbiotica </em>electrocompetent: </strong>(protocol based on <em>E. coli </em>version: [[ProtocolsElectrocompetentCells][http://barricklab.org/twiki/bin/view/Lab/ProtocolsElectrocompetentCells]]<em>)</em>
   1 Grow up <em style="background-color: transparent">S. symbiotica </em><span style="background-color: transparent">for two days from glycerol stock at room temperature (shaking)</span>
   1 Innoculate 500ul 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 (the longer the 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*

   * 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!

-- Main.KateElston - 28 Apr 2017
Barrick Lab > ProtocolList > ProtocolsWorkingWithSerratiaSymbiotica
Topic revision: r2 - 2017-05-09 - 18:17:21 - Main.KateElston