Nissle 1917 (EcN) E.coli strain益生菌大肠杆菌菌株 BioVector NTCC质粒载体菌种细胞基因保藏中心

Nissle 1917 (EcN) E.coli strain益生菌大肠杆菌菌株 BioVector NTCC质粒载体菌种细胞基因保藏中心                      

The E. coli strain Nissle 1917 (EcN) disposes of a number of properties which are important to its survival, colonization ability, and persistence in the intestinal ecosystem, and to its therapeutic efficacy.
• Mobility due to type H1-flagella
• Ability to colonize due to specific interaction of its ¬flagellae with the mucin layer
  Ability to form biofilms due to F1A-, F1C- and Curli-fimbriae
• Antagonistic activity directed against foreign microbes due to antimicrobial microcins
   Vitality and resilience (“biological fitness”) due to ¬multiple iron uptake systems
• Immunomodulatory effects due to strain-specific ¬surface structures (special LPS = lipopolysaccharide) and synthesis of immunomodulatory signal substances
• Anti-inflammatory effects
• Stabilizing effect on the barrier function of the intestinal mucosa
References
1) Blum G et al. Properties of Escherichia coli strains of serotype O 6. Infection 1995; 23: 234–236.
2) Sonnenborn U, Schulze J. The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic. Microbial Ecol Health Dis 2009; 21: 122–158.
3) Schiemann M et al. 125 Jahre E. coli – Bedeutung in Forschung und Medizin. Alfred-Nissle¬-Gesellschaft (Hrsg.) Hagen, ISBN 3-9811198-4-3, 2010.
Recovery
1. Obtain an LB agar plate with the appropriate antibiotic.
2. Using a sterile pipette tip, touch the bacteria growing within the punctured area of the stab culture. (A sterilized wire loop or sterile toothpick can be used in place of a sterile pipette tip.)
3. Run this tip lightly over a section of the plate, as shown in the figure, to create streak #1.
4. Using another sterile pipette tip, pass through streak #1 and spread the bacteria over a second section of the plate, to create streak #2.
5. Using a third sterile pipette tip, pass through streak #2 and spread the bacteria over the last section of the plate, to create streak #3.
6. Grow overnight in a 37 o C incubator (unless a different growth temperature is indicated on the plasmid datasheet).
7. In the morning, single colonies should be visible. If the bacterial growth is too dense, re-streak onto a new agar plate to obtain single colonies.
Culture of E. coli Nissle 1917 (EcN)
Protocol:
1. Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle:
o 4 g NaCl
o 4 g Tryptone
o 2 g Yeast Extract
o and dH2O to 400 mL
Note: If your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above.
Media without growth (top) and with growth (bottom)


Loosely close the cap on the bottle (do NOT close all the way or the bottle may explode!) and then loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to cool to room temperature. Now screw on the top of the bottle and store the LB at room temperature.
2. When ready to grow your culture, add liquid LB to a tube or flask.
3. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
4. Drop the tip or toothpick into the liquid LB and swirl.
5. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
6. Incubate bacterial culture at 37°C for 12-18 hr in a shaking incubator.
7. After incubation, check for growth, which is characterized by a cloudy haze in the media (see right).
8.  (Optional) For long term storage of the bacteria, you can proceed with Creating a Glycerol Stock.
Creating Bacterial Glycerol Stocks for Long-term Storage
Bacteria on an LB agar plate can be stored at 4°C for a few weeks. However, if you want to store bacteria for a longer time, you will need to establish glycerol stocks. The addition of glycerol stabilizes the frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock of bacteria can be stored stably at -80°C for many years.
Protocol
1. Follow the steps for Inoculating an Overnight Liquid Culture.
2. After you have bacterial growth, add 500 μL of the overnight culture to 500 μL of 50% glycerol in a 2 mL screw top tube or cryovial and gently mix.
Note: Make the 50% glycerol solution by diluting 100% glycerol in dH20.
Note: Snap top tubes are not recommended as they can open unexpectedly at -80°C.
3. Freeze the glycerol stock tube at -80°C. The stock is now stable for years, as long as it is kept at -80°C. Subsequent freeze and thaw cycles reduce shelf life.
4. To recover bacteria from your glycerol stock, open the tube and use a sterile loop, toothpick or pipette tip to scrape some of the frozen bacteria off of the top. Do not let the glycerol stock unthaw! Streak the bacteria onto an LB agar plate.
5. Grow your bacteria overnight at 37°C.
Production of Chemically Competent E. coli Nissle 1917 (EcN) Cells
1. Inoculate one colony from LB plate into 2 ml LB liquid medium. Shake at 37 °C overnight.
2. Inoculate 1-ml overnight cell culture into 100 ml LB medium (in a 500 ml flask).
3. Shake vigorously at 37 °C to OD600 ~0.25-0.3.
4. Chill the culture on ice for 15 min. Also make sure the 0.1M CaCl2 solution and 0.1M CaCl2 plus 15% glycerol are on ice.
5. Centrifuge the cells for 10 min at 5000 g at 4°C.
6. Discard the medium and resuspend the cell pellet in 30-40 ml cold 0.1M CaCl2. Keep the cells on ice for 30 min.
7. Centrifuge the cells as above.
8. Remove the supernatant, and resuspend the cell pellet in 6 ml 0.1 M CaCl2 solution plus 15% glycerol.
9. Pipet 0.4-0.5 ml of the cell suspension into sterile 1.5 ml micro-centrifuge tubes. Flash freeze these tubes in liquid nitrogen and then transfer them to the -80 C freezer.
 Note: Successful transformations have occured with 100uL of cells + 1ug of DNA, however the efficency of cells made through this process is lower than that of Subcloning Efficency DH5a from Invitrogen. After flash freezing, competency of cells prepared through this protocol increases over time with additional storage time in -80°C for approximately 3 days.
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