---+ 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<span style="background-color: transparent">Innoculate 500ul culture into 50ml TSB in 250ml flask</span> 1 Grow until OD ~ 0.4 (check after 16 hours)<span style="background-color: transparent">Grow until OD ~ 0.4 (check after 16 hours)</span> 1 Transfer 25 ml into two Falcon tubes<span style="background-color: transparent">Transfer 25 ml into two Falcon tubes</span> 1 Centrifuge at 6000rpm for 5min at 4°C<span style="background-color: transparent">Centrifuge at 6000rpm for 5min at 4°C</span> 1 Wash with 20 ml 10% glycerol<span style="background-color: transparent">Wash with 20 ml 10% glycerol</span> 1 Repeat wash step 4 more times<span style="background-color: transparent">Repeat wash step 4 more times</span> 1 Resuspend in 250ul 10% glycerol<span style="background-color: transparent">Resuspend in 250ul 10% glycerol</span> 1 Divide into 50ul aliquots and freeze at -80 or continue electroporation protocol<span style="background-color: transparent">Divide into 50ul aliquots and freeze at -80 or continue electroporation protocol</span> *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
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Topic revision: r1 - 2017-04-28 - KateElston