IGEM:Harvard/2006/Cyanobacteria/Protocols: Difference between revisions

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==Zero-blunt TOPO cloning kit==
==Zero-blunt TOPO cloning kit==
*This kit is used for cloning blunt-ended PCR products, such as those produced by Vent polymerase. See the protocol at Invitrogen's website [http://www.invitrogen.com/content/sfs/manuals/zeroblunttopo_man.pdf here].
*This kit is used for cloning blunt-ended PCR products, such as those produced by Vent polymerase. See the protocol at Invitrogen's website [http://www.invitrogen.com/content/sfs/manuals/zeroblunttopo_man.pdf here].
==Midiprep Protocol==
Key: Low-Copy (LC), High-Copy (HC)
Pre-Midiprep
:# Put buffer P3 in the refrigerator.
:# Check buffer P2 for precipitate; if there is then redissolve @ 37°C.
Growth
:# Pick a single colony and inoculate a sulture of 2-5 ml LB, incubate for ~8 h at 37°C with shaking.
:# Dilute the starter culture 1/500 to 1/1000 into selective LB medium.
:# High-copy: 25 mL; Low-copy: 25 mL.
Midiprep
:# Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
:#* Big centrifuge: SLA-1500; Rotor code 28; 6290 rpm.
:#* Big centrifuge: HB6; Rotor code 23; 6060 rpm.
:# Resuspend the bacterial pellet in 4 ml(HC) or 10 ml Buffer P1(LC).
:# Add 4 ml(HC) or 10 ml(LC) Buffer P2, mix by inverting 4-6 times, then incubate at room temperature for <5 minutes.
:# Add 4 ml(HC) or 10 ml(LC) Buffer P3, mix by inverting 4-6 times, then incubate on ice for 15-20 minutes.
:# Mix the sample again.
:# Centrifuge at >20,000 x g (or 12,000 rpm) for 30 min at 4&deg;C, and remove the supernatant promptly.
:#* Centrifuge in non-glass tube
:# Centrifuge the supernatant again at >20,000 x g for 15 min at 4&deg;C
Professor George Church: Entrapment


== References ==
== References ==

Revision as of 12:37, 14 August 2006

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Media

1L agar for freshwater cyanobacteria (no glucose)

This protocol produces 1L agar suitable for plating freshwater cyanobacteria (PCC7942 and PCC6803). [1]

  1. Mix 10 g agar and 1 mM thiosulfate (= 0.248 g), top off with H20 to 500 mL total volume.
  2. Autoclave the product of (1)
  3. Mix 20mL 50x BG-11 solution and 480 mL of H20
  4. Autoclave the product of (3)
  5. Mix (2) and (4), pour plates and let cool

1L agar for freshwater cyanobacteria (with glucose)

This protocol produces 1L agar with 5 mM glucose suitable for plating PCC6803 cyanobacteria.

  1. Mix 10 g agar and 1mM thiosulfate (= 0.248 g), top off with H20 to 500 mL total volume
  2. Autoclave the product of (1)
  3. Mix 20 mL 50x BG-11 solution and 230 mL of H20
  4. Autoclave the product of (3)
  5. Mix 5 mL glucose and 245 mL of H20. [2]
  6. Autoclave the product of (5)
  7. Mix (2) and (4) and (6), pour plates and let cool

1L liquid media for freshwater cyanobacteria

This protocol produces 1L of liquid media suitable for culturing PCC7942 and PCC6803.

  1. Add 20 mL 50x BG-11 and 1 mM thiosulfate (= 0.248 g), top off with H20 to 1L total volume
  2. Filter the solution into the desired container with a 0.2 µm filter.

Notes on growing freshwater cyanobacteria in liquid media

  • Spray your gloves, containers, and working surfaces with ethanol, and let the ethanol dry, before handling cyanobacteria. We take this precaution because our bacteria do not have any antibiotic resistance as far as we know, so contamination is a bigger risk than with E. coli.
  • The algae guide recommends growing bacteria in 125mL of media in a 250 mL Erlenmeyer flask (to give an idea of how high the flask should be filled).
  • Use notched caps on the flasks, to permit gas flow while avoiding aerial contamination.
  • When inoculating a culture, use autoclaved toothpicks to scrape the colony/streaks of interest, and throw them in the solution.
  • According to Jeffrey Chabot's PhD thesis, cyanobacteria should be grown at 4200lux, cool white fluorescent lighting; this illumination can be higher during the initial growth stage.

Freezing and thawing freshwater cyanobacteria

Freezing for freshwater strains

  1. Run the bacteria through a centrifuge to form a pellet. 10X concentration, pellet down at least 5 minutes!
  2. Resuspend pullet in 8% DMSO solution / 46% 1x BG-11 / 46% H20, in a cryo-friendly tube
  3. Freeze (-70C or -80C is fine)

Thawing

  1. After taking the bacteria out of the freezer, pass them briefly through a stream of hot water or rub them with your fingers. This melts the solution around the walls of the tube.
  2. Open the tube and jab the center of the frozen mass with a sterile toothpick, then pull the mass out, like pulling a popsicle out of a popsicle mold.
  3. Immediately place the frozen bacteria in liquid media. Give a week or so for the bacteria to recover and regrow.

Transformation in cyanobacteria

Zero-blunt TOPO cloning kit

  • This kit is used for cloning blunt-ended PCR products, such as those produced by Vent polymerase. See the protocol at Invitrogen's website here.

Midiprep Protocol

Key: Low-Copy (LC), High-Copy (HC)

Pre-Midiprep

  1. Put buffer P3 in the refrigerator.
  2. Check buffer P2 for precipitate; if there is then redissolve @ 37°C.

Growth

  1. Pick a single colony and inoculate a sulture of 2-5 ml LB, incubate for ~8 h at 37°C with shaking.
  2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium.
  3. High-copy: 25 mL; Low-copy: 25 mL.

Midiprep

  1. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
    • Big centrifuge: SLA-1500; Rotor code 28; 6290 rpm.
    • Big centrifuge: HB6; Rotor code 23; 6060 rpm.
  2. Resuspend the bacterial pellet in 4 ml(HC) or 10 ml Buffer P1(LC).
  3. Add 4 ml(HC) or 10 ml(LC) Buffer P2, mix by inverting 4-6 times, then incubate at room temperature for <5 minutes.
  4. Add 4 ml(HC) or 10 ml(LC) Buffer P3, mix by inverting 4-6 times, then incubate on ice for 15-20 minutes.
  5. Mix the sample again.
  6. Centrifuge at >20,000 x g (or 12,000 rpm) for 30 min at 4°C, and remove the supernatant promptly.
    • Centrifuge in non-glass tube
  7. Centrifuge the supernatant again at >20,000 x g for 15 min at 4°C

Professor George Church: Entrapment

References


[1]: The use of thiosufate is discussed in the following papers; it is isn't required, but it supposedly improves growth rate.

  1. Thiel T, Bramble J, and Rogers S. Optimum conditions for growth of cyanobacteria on solid media. FEMS Microbiol Lett. 1989 Oct 1;52(1-2):27-31. DOI:10.1016/0378-1097(89)90164-x | PubMed ID:2513249 | HubMed [cyano_prot1]
  2. Ohkawa H, Price GD, Badger MR, and Ogawa T. Mutation of ndh genes leads to inhibition of CO(2) uptake rather than HCO(3)(-) uptake in Synechocystis sp. strain PCC 6803. J Bacteriol. 2000 May;182(9):2591-6. DOI:10.1128/JB.182.9.2591-2596.2000 | PubMed ID:10762263 | HubMed [cyano_prot2]

All Medline abstracts: PubMed | HubMed


[2]: Usage of glucose is to take advantage of heterotrophic growth of PCC6803 with glucose; this supposedly cuts down the growth cycle to 4 days, according to the 2006 MIT iGEM team.

  1. Spence E, Bailey S, Nenninger A, Møller SG, and Robinson C. A homolog of Albino3/OxaI is essential for thylakoid biogenesis in the cyanobacterium Synechocystis sp. PCC6803. J Biol Chem. 2004 Dec 31;279(53):55792-800. DOI:10.1074/jbc.M411041200 | PubMed ID:15498761 | HubMed [cyano_prot3]


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