Faster engineering for cyanobacteria

Developing a series of plasmids and a method to rapidly increase production of cyanobacterial mutants with multiple alterations.

The Idea

Cyanobacteria (oxygenic photosynthetic bacteria) are evolutionary ancient organisms and significant primary producers found in almost every environment on Earth. Several species, including the genetically tractable Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis), are used as model systems to study both photosynthesis and cyanobacterial metabolism and physiology. Synechocystis is also increasingly being considered for chemical and biomass production due to their highly efficient conversion of water and CO2 to biomass using solar energy and growth on non-arable land with minimal nutrients.

We have already developed a series of tools that allow us to generate ‘unmarked’ mutants in Synechocystis, that is genetically modified strains containing no foreign DNA, unless when desired. To generate mutant strains, plasmids containing two fragments identical to regions in the cyanobacteria chromosome flanking the gene to be deleted (termed the upstream and downstream flanking regions are first constructed. Two genes are then inserted between these flanking regions. One of these encodes an antibiotic resistance protein, the second SacB. In the first stage of the process, marked mutants are generated. The plasmid construct is mixed with Synechocystis and the DNA is naturally taken up by the cell. Transformants are selected by growth on agar plates containing the appropriate antibiotic and the mutant genotype verified by PCR. To generate unmarked mutants, the marked mutant is then mixed with a second plasmid containing just the flanking regions or the flanking regions with an expression cassette between the inserts. Selection is via growth on agar plates containing sucrose. As sucrose is lethal to cells when the sacB gene product is expressed, only cells in which a second recombination event occurs, whereby the sucrose sensitivity gene, in addition to the antibiotic resistance gene are recombined out of the chromosome and onto the plasmid, will survive. In exchange the flanking regions and when applicable the DNA between them is inserted into the chromosome.

There are a number of significant advantages to generating unmarked mutants. Because the incoming DNA has been removed in the unmarked mutant, the entire process can be repeated multiple times in the same strain. Therefore it is possible to make as many alterations to a strain as desired. In addition, the absence of foreign DNA, particularly genes encoding antibiotic resistance proteins, in the mutated strain is desirable as it avoids the possibility of ‘escape’ of organisms containing foreign genes into the environment. The one great disadvantage is the time in which it takes to generate a mutant strain, typically 5-6 weeks. The purpose of this project is to develop a series of plasmids and a method which will rapidly increase the speed at which cyanobacterial mutants with multiple alterations are constructed.