ProtocolsCulturingSnodgrassellaAlvi < Lab

---++!! Working with <i>Snodgrassella alvi</i>

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_Snodgrassella alvi_ is a member of the honey bee (_Apis mellifera_) gut core microbiota that can be cultivated _in vitro_. Its isolation and characterization were recently carried out by [[https://wkkwong.com][Waldan Kwong]] in the laboratory of [[http://web.biosci.utexas.edu/moran/][Dr. Nancy Moran]] and are described in depth in [[http://ijs.microbiologyresearch.org/content/journal/ijsem/10.1099/ijs.0.044875-0][here]].

---+++ *In vitro Culture Conditions*

---++++ Growth in Solid Media/Agar Plates
 _Snodgrassella alvi_ strain wkB2 (type strain) is generally cultured at 35°C and 5% CO<sub>2</sub>. It grows robustly on agar plates after 48 hours on the following media: 
   * Brain Heart Infusion Agar (BHI)
   * Brain Heart Infusion Agar (BHI) supplement with 5% Sheep's Blood
   * Columbia Agar supplement with 5% Sheep's Blood (CBA) - *PREFERRED*
   * R2 Agar

---++++ Liquid Media
 _Snodgrassella alvi_ can be cultivated in the following liquid media (same general culture conditions as above, with or without shaking): 
   * Insectagro (proprietary media)
   * SFM4Insect with L-Glutamine (proprietary media)
   * Salt-free LB
   * Columbia broth
   * R2 Liquid Media
   * Brain Heart Infusion (BHI) media

Insectagro and SFM4Insect media induces maximum biofilm formation in _S. alvi_ cultures.

Recently, Philip Engel's group published on using a modified version of M9 minimal media called the *Bee9 medium* to be able to robustly culture _S. alvi_ in liquid (Quin _et al_ 2024).

---+++ *Transformation*

---++++ Conjugation
 _Snodgrassella alvi_ can be conjugated using _E. coli_ donor strains and mobilizable rsf1010-based plasmids. Refer to the Bee Microbiome Toolkit paper for full description of working with this species. <Br>
Perform conjugation as described here: [[ProtocolsConjugation][Conjugation]], with the following specifics: use 10:1 ratio of _S. alvi_ to donor _E. coli_ pooled on CBA plates. Incubate overnight, then resuspend and plate on selective CBA plates.

---++++

---++++ Electroporation

   * *Electrocompetent Cell Preparation:*

_S. alvi_ can be transformed by electroporation (Chhun _et al_ 2024; Lariviere _et al_ 2023). Briefly, streak wkB2 cells using overlapping streaks on CBA and grow at 35°C in 5% CO<sub>2</sub> for 2-3 days (recommended 72 hours). Once colonies have appeared, pick colonies, re-streak multiple CBA plates, and incubate them for 24-48 hours (not exceeding 48 hours) in the same condition. Scrape all the growth from the plate into 1.5 ml tube(s) with 1 ml PBS and resuspend/mix cells, and pellet by centrifuging at 5000 RCF (xg) for 10 mins. Mix the cells in the tube by pipetting up and down. Wash twice in 1 ml chilled autoclaved DI water or 10% glycerol and resuspend the pellet after the final wash in 10% glycerol. Keep the cells on ice from here on out. Make 70-100 µl aliquots and either freeze at -80°C or use right away.

   * *Electroporation:*

To electroporate _S. alvi_ (Chhun _et al_ 2024; Lariviere _et al_ 2023), obtain and thaw an aliquot of electrocompetent wkB2 cells and add the appropriate amount of DNA (0.5-1 µg recommended if using RSF1010-based plasmids). After adding plasmid DNA, incubate the cells  on ice for 20 minutes. Afterwards, transfer to a 1 mm electroporation cuvette and electroporate (Ec1 on BioRad _Micropulser_). Immediately following electroporation, add 900 µl of SFM4 media or Columbia broth to the cuvette and transfer the cells to a 50 ml falcon tube, with the cap loosely taped on (to allow for gas exchange). Allow cells to recover overnight at 35°C in 5% CO<sub>2</sub> for 2-3 days. The next day, plate 100 µl of undiluted cells on selective media. Pellet the remainder of the cells, resuspend in 100 µl of media, and plate the concentrate on selective media as well. Grow selective plates at 35°C in 5% CO<sub>2</sub> for 2-3 days or until colonies form. Once colonies have formed, make an isolation streak on a new plate and grow at 35°C in 5% CO<sub>2</sub> for 2-3 days. Once isolation streaks have grown up, make glycerol stocks and store at -80°C.

   * *Genomic Integration by Electroporation:*

To insert genes directly into _S. alvi_ genome by electroporation, (Lariviere _et al_ 2023), below is the recommended DNA concentrations based on the type of DNA:
   * Gene fragment/Plasmid - 1 µg
   * Purified Genomic DNA - 2 µg


---+++ *Antibiotic Concentrations and Stock Solutions*

This table lists “standard” concentrations of antibiotics commonly used in microbiology laboratories. These concentrations apply to _S. alvi_ (wkB2). Antibiotic selection in other organisms may use different concentrations, so check primary literature for more info.  
%TABLE{headeralign="left,center,center,center,center,left" dataalign="left,center,center,center,center,left,center,left"}%
| *Reagent* | *Abb* | *Stock Conc.* | *Working Conc.* | *Dilution* | *Solvent* | *MSDS* | *Notes* |
| Ampicillin | Amp | 100 mg/mL | 20 µg/mL | 5,000× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=A9518&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fa9518%3Flang%3Den][ link]] | Ampicillin degrades quickly in both plates and stock solutions. Culture plates with Amp can be stored at 4°C for about 2 weeks. Stock solutions can be stored at 4°C for 2 weeks but can last as long as 4-6 months when stored at -20°C. In general, use carbenicillin for ampicillin selection, because it is more chemically stable. |
| Carbenicillin | Crb | 100 mg/mL | 50 µg/mL | 2,000× | 50% EtOH | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=C1389&brand=SIAL&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsial%2Fc1389%3Flang%3Den][link]] | Carbenicillin can be used in place of Ampicillin at the same working concentration. Carbenicillin is more stable than Ampicillin, and is generally preferable. |
| Chloramphenicol | Cam | 20 mg/mL | 20 µg/mL | 1,000× | EtOH | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=C0378&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fc0378%3Flang%3Den][link]] | |
| Gentamicin | Gen | 20 mg/mL | 25 µg/mL | 800× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927175][link]] | For non-canonical experiments: <br />Use 20 µg/mL in culture from 20mg/mL 1000× stock |
| Kanamycin | Kan | 50 mg/mL | 25 µg/mL | 2,500× |  ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927199][link]] | Plates start to lose sensitivity after 2 weeks; [[https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/2/k1377pis.pdf][recommended]] Kanamycin plates only last a few weeks at 4°C; best to make fresh Kan plates every 2 - 4 weeks. |
| Nalidixic Acid | Nal | 30 mg/mL | 30 µg/mL | 1,000× |  1M NaOH | [[http://www.sciencelab.com/msds.php?msdsId=9926169][link]] | |
| Rifampicin | Rif | 50 mg/mL | 30 µg/mL | 1667× | MeOH + drops of 10M NaOH |  [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=R3501&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fr3501%3Flang%3Den][link]] |  Toxic to humans, use PPE! Rifampicin is TOXIC to humans, light-sensitive, and degrades in solution rather quickly. It needs to be stored wrapped in foil and replaced every 2 months. |
| Spectinomycin | Spec | 60 mg/mL | 30 µg/mL | 2,000× | ddH<sub>2</sub>O | [[http://www.sigmaaldrich.com/MSDS/MSDS/DisplayMSDSPage.do?country=US&language=en&productNumber=S4014&brand=SIGMA&PageToGoToURL=http%3A%2F%2Fwww.sigmaaldrich.com%2Fcatalog%2Fproduct%2Fsigma%2Fs4014%3Flang%3Den][link]] |
| Streptomycin | Str | 100 mg/mL | 20 µg/mL | 5,000× | ddH<sub>2</sub>O | [[http://www.sciencelab.com/msds.php?msdsId=9927414][link]] | |
| Tetracycline | Tet | 10 mg/mL | 10 µg/mL | 1,000× | ddH<sub>2</sub>O | [[https://www.fishersci.com/store/msds?partNumber=AC233100025&productDescription=TETRACYCLINE+HYDROCHLORI+2.5GR&vendorId=VN00032119&countryCode=US&language=en][link]] | _S. alvi_ is *naturally resistant* to Tet. Use 10 µg/mL. Can be used to isolate _S. alvi_ from other bacteria or contaminants during bee colonization assays without other antibiotics. |
| Trimethoprim | TMP | 10 mg/mL | 20 µg/mL | 500× | DMSO | [[https://www.fishersci.com/store/msds?partNumber=AC455120050&productDescription=TRIMETHOPRIM%2C+98%25+5GR&vendorId=VN00032119&countryCode=US&language=en][link]] | _S. alvi_ is *naturally resistant* to TMP. Use 20 µg/mL. |
| Tylosine | Tyl | 5 mg/mL | 5 µg/mL | 1,000× | ddH<sub>2</sub>O | [[https://www.fishersci.com/store/msds?partNumber=AC463070050&productDescription=TYLOSIN+TARTRATE+5GR&vendorId=VN00032119&countryCode=US&language=en][link]] | _S. alvi_ wkB2 and _S. communis_ wkB12 are *naturally resistant* to Tyl. Use 5 µg/mL. |

*References:*

   * Kwong WK, Moran NA. Cultivation and characterization of the gut symbionts of honey bees and bumble bees: description of Snodgrassella alvi gen. nov., sp. nov., a member of the family Neisseriaceae of the Betaproteobacteria, and Gilliamella apicola gen. nov., sp. nov., a member of Orbaceae fam. nov., Orbales ord. nov., a sister taxon to the order 'Enterobacteriales' of the Gammaproteobacteria. Int J Syst Evol Microbiol. 2013 Jun;63(Pt 6):2008-2018. doi: 10.1099/ijs.0.044875-0. Epub 2012 Oct 5. PMID: 23041637.

   * Leonard SP, Perutka J, Powell JE, Geng P, Richhart DD, Byrom M, Kar S, Davies BW, Ellington AD, Moran NA, Barrick JE. Genetic Engineering of Bee Gut Microbiome Bacteria with a Toolkit for Modular Assembly of Broad-Host-Range Plasmids. ACS Synth Biol. 2018 May 18;7(5):1279-1290. doi: 10.1021/acssynbio.7b00399. Epub 2018 Apr 13. PMID: 29608282; PMCID: PMC5963681.

   * Quinn, A., El Chazli, Y., Escrig, S. et al. Host-derived organic acids enable gut colonization of the honey bee symbiont Snodgrassella alvi. Nat Microbiol 9, 477–489 (2024). https://doi.org/10.1038/s41564-023-01572-y.

   * Lariviere PJ, Leonard SP, Horak RD, Powell JE, Barrick JE. Honey bee functional genomics using symbiont-mediated RNAi. Nat Protoc. 2023 Mar;18(3):902-928. doi: 10.1038/s41596-022-00778-4. Epub 2022 Dec 2. PMID: 36460809.

   * Chhun A, Moriano-Gutierrez S, Zoppi F, Cabirol A, Engel P, Schaerli Y. An engineered bacterial symbiont allows noninvasive biosensing of the honey bee gut environment. PLoS Biol. 2024 Mar 5;22(3):e3002523. doi: 10.1371/journal.pbio.3002523. PMID: 38442124; PMCID: PMC10914260.

   * Single-step genome engineering in the bee gut symbiont Snodgrassella alvi. Patrick J. Lariviere, A. H. M. Zuberi Ashraf, Sean P. Leonard, Laurel G. Miller, Nancy A. Moran, Jeffrey E. Barrick. bioRxiv 2023.09.19.558440; doi: https://doi.org/10.1101/2023.09.19.558440
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Topic revision: r12 - 2025-03-20 - ZuberiAshraf