---+ 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 <figure> <img style="border-color: white; border-style: solid; border-width: 10px;" src="%ATTACHURLPATH%/normal_Ss.JPG" alt="normal_Ss.JPG" width="210.75" height="235.5" align="right" > </figure> *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 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; *<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}%
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Topic revision: r8 - 2022-08-11 - KateElston