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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<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" >
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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!

*Using Narishige IM-400 to inject _S. symbiotica_ into aphids*

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   1 Plan your injections accordingly. Remember  <i>S. symbiotica</i> takes twice as long as <i>E. coli</i> to reach saturation
   1 Spin down saturated culture of S. symbiotica at 6800 x g for 3 minutes
   1 Wash 3 times in 1X PBS, spinning down at 6800 x g for 3 minutes
   1 Resuspend in buffer A (25 mM KCl, 10 mM MgCl2, 250 mM sucrose, 35 mM Tris-HCl, pH 7.5) and normalize to OD600 of 1
   1 Harvest 4th instar (7 day-old) aphids and place them on ice -- this prevents them from moving around when you eventually go to inject
   1 When ready to inject, turn on the microscope (Leica), the vacuum aphid holder, and open the valve for the compressed air cylinder.
   1 Flip the switch on the back of the IM-400 injector -- the parameters panel should read the following: injection: 0.020 MPa, 0.20 sec, balance ON, 0.002 MPa.
   1 Attach your pulled capillary tube needle to the injector -- using a pair of forceps, break the tip of the pulled needle to "open" it. 
   1 To pull up liquid into the tube, press and hold down the SUC button on the parameter controller while the tip of the needle is submerged in your tube containing the OD600 of 1 resuspension of <i>S. symbiotica</i>
   1 Once you have pulled up enough culture, press the right pedal while looking at the tip of the needle under the microscope -- a small amount of liquid should be discharged (<0.1 uL). The drop should be almost invisible to the naked 
      eye. This insures that you are injecting ~1000 cells with the given considering the OD600 of 1.0 that the cells were resuspended to.
   1 With the needle loaded, place an aphid onto the vacuum suction holder. The back of the aphid should be attached to the pipette tip of the holder
   1 While looking under the microscope, use the joystick to manipulate needle so that it clearly pierces the skin of the aphid directly behind where the third leg connects to the abdomen.
   1 Press the right pedal to discharge the cells
   1 Remove the injected aphid from the holder and place it into a petri dish with a Kim wipe covering the floor of the petri dish. The aphid will lose a little hemolymph which the Kim wipe will absorb. If you do not add the Kim wipe, the 
      leaking hemolymph will cause the aphid to stick to the floor of the petri dish resulting in almost certain death.
   1 Place a new aphid on the pipette tip of the vacuum holder and repeat this process. You should inject, at minimum, 40 aphids per injection treatment. Several aphids will die from their wounds within the first 48 hours post-injection. In 
      order to still have a large enough sample to see how the bacteria is affecting aphid survival, a minimum of 40 aphids is required.
   1 If you are injecting more than one treatment, you need to either wash the pulled capillary needle or use a new pulled capillary needle. To wash the needle, discharge the remainder of the prior treatment from the needle by holding 
      down the left pedal.
   1 Dip the needle into a tube of 70% ethanol and hold down the SUC button as the ethanol is pulled into the tube. Discharge using left pedal. Then dip the needle into buffer A. Repeat the pulling up and discharging process. Then, insert 
      the cleaned needle into the next treatment of resuspended bacteria and repeat the injection process.




-- %USERSIG{KateElston - 2022-08-11}%%USERSIG{AnthonyVandieren - 2025-03-20}%
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I	Attachment	History	Action	Size	Date	Who	Comment
jpg	20250319_145959.jpg	r1	manage	4152.2 K	2025-03-20 - 21:19	AnthonyVandieren	Narishige IM-400 microinjector setup
This topic: Lab > WebHome > ProtocolList > ProtocolsWorkingWithSerratiaSymbiotica
Topic revision: r9 - 2025-03-20 - AnthonyVandieren