Cyano Q&A
A lot of current iGEM teams work with cyanobacteria. These tiny green microorganisms require special cultivation methods which aren’t always known to everyone. We started noting down important facts after iGEM Hamburg 2018 gave us the impetus to do so. We created this Cyano Q&A in order to solve the most prominent questions of them and other teams that contacted us. But it is also written for everyone else who is entering the world of cyanobacteria.
What are the cultivation conditions of cyanobacteria?
That depends on the strain. Typically Synechococcus elongatus sp 7942 and Synechocystis PCC 6803 - two model organisms - are incubated at 30°C (86°F). Moreover, as an autotrophic organism they need light in order to survive. We use light bulbs with a light intensity of 75 μE/m2·s . With those settings they have a growth rate of 0.052 h‐1. Optionally, you can use the light intensity of 60 μE/m2·s. With those adjustment the growth rate would be 0.026 h‐1.
Can I store cyanobacteria on agar plates and if so, how?
Yes, you can store them on plates made of equal amounts of 1,5% agar and 2x BG11. You can also add an antibiotic treatment if needed. You can find the protocol for this here: https://www.protocols.io/view/recipe-for-standard-bg-11-media-gtmbwk6
Are cyanobacteria transformable?
Yes! You can use conjugation, natural transformation or even electroporation for gene transfer. In our project we used the conjugation method. The protocol can be found here: https://www.protocols.io/view/triparental-mating-of-synechocystis-ftpbnmn
What kind of plasmids are needed for transformation?
Normally cyanobacteria are transformed using genomic integration. Therefore a plasmid should have fragments which overlap with neutral sites in the genome of the chassis organism. For example you can use NSIa and NSIb for this purpose. It is also possible to transfer plasmids using triparental mating.
How do you gain competent cyanobacteria?
Many cyanobacteria, like Synechococcus elongatus PCC 7942 or Synechocystis PCC 6803 are naturally competent. There are also transformation kits available for the transformation.
Are cyanobacteria compatible with E. coli biobricks?
It depends. Some biobricks like the ribosome binding site BB_0034 are compatible. Generally you can use them, but they may work less efficiently than in Escherichia coli.
Which medium is needed in order to cultivate cyanobacteria?
It depends on the strain because some strains need freshwater media and others need saltwater based media. The usually used medium for freshwater strains like Synechococcus elongatus is called BG11. The protocol for the preparation of BG11 is listed here: BG11. For marine strains the same medium can be used with additional salt.
Are there any toxic metabolites released by cyanobacteria?
It is possible for S. elongatus to produce microcystin and β-Methylamino-L-alanine, but it is rarely released into the media and can be neglected.
How long is it possible to cultivate cyanobacteria?
Basically forever as long as the medium is frequently refreshed and there is no contamination.
What is the typical yield of sugar production of cyanobacteria?
There is little production of sucrose from the wild type. Some modified strains like the S. elongatus sp. PCC 7942 cscB strain from Daniel Ducat et al., 2011 have a production rate of 36.1 mg / liter * h.
At which wavelength do you measure cyanobacteria?
When you want to measure the OD of your culture, use 750 nm. If you want to measure the autofluorescence you have to excite at 620 nm. Afterwards the fluorescence emission can be measured at 680 nm.