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 [3, 4]. 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 56 weeks. This is due to the relatively slow growth of the organism. 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.
 D.C. Ducat, J.C. Way, P.A. Silver, Engineering cyanobacteria to generate highvalue products, Trends Biotechnol 29 (2011) 95103.
 G.C. Dismukes, D. Carrieri, N. Bennette, G.M. Ananyev, M.C. Posewitz, Aquatic phototrophs: efficient alternatives to landbased crops for biofuels, Current Opinion in Biotechnology 19 (2008) 235 240.
 D.J. LeaSmith, N. Ross, M. Zori, D.S. Bendall, J.S. Dennis, S.A. Scott, A.G. Smith, C.J. Howe, Thylakoid terminal oxidases are essential for the cyanobacterium Synechocystis sp. PCC 6803 to survive rapidly changing light intensities, Plant Physiol 162 (2013) 484495.
 D.J. LeaSmith, P. Bombelli, J.S. Dennis, S.A. Scott, A.G. Smith, C.J. Howe, Phycobilisome-Deficient Strains of Synechocystis sp. PCC 6803 Have Reduced Size and Require CarbonLimiting Conditions to Exhibit Enhanced Productivity, Plant Physiol 165 (2014) 705714.
Who we are:
David LeaSmith. Postdoctoral associate in the Department of Biochemistry (Chris Howe lab). I specialise in microbial genetics and biochemistry with a focus on photosynthetic organisms (cyanobacteria and purple bacteria). I use synthetic biology, genomics and genetic tools to understand bacterial physiology and metabolism and develop strains with increase biofuel or electrical output using energy derived from either photosynthesis or degradation of waste products.
I will be the sole person working on this project since outside collaborators are not required to complete it.
We have already generated plasmids and methods for genetic manipulation of Synechocystis which have been distributed to a range of laboratories, both in the U.K. and overseas. Following a recent publication, I was contacted by the environmental editor of the Journal of Visualised Experiments (JOVE) who offered to publish our research methods. JOVE presents methods in a video format, allowing each step to be detailed visually, which is extremely useful for complex and technically difficult protocols. The publication fee and filming costs is £1600 which I would like to cover with this grant since my current funding is focused on a different area and cannot be used to publish this work. I believe this fits into the focus of this study, since the methods we have already developed are similar to what is detailed in this proposal. The overall aim of this project is to develop a range of plasmids, test these in Synechocystis and generate mutant strains with multiple chromosomal alterations. If development of the method is successful the first goal would be to publish this research in a peer reviewed journal. Following publication the plasmids and method would be freely available to other research groups. The first stage of this process is to construct a series of four plasmids, each containing the sacB gene but a different antibiotic resistance cassette (kanamycin, apramycin, spectinomycin and erthyromycin). The plasmid with the kanamycin resistance cassette is already constructed and has been used in our previous work. Each of these plasmids will be generated by Gibson cloning and will be flanked by appropriate restriction enzyme sites, which will allow insertion between various flanking regions. These cassettes will be inserted into different plasmids with flanking regions targeting nonessential genes. These plasmids have already been constructed. Following completion, 1, 2, 3 and 4 plasmids will be transformed into Synechocystis. Due to its ability to naturally uptake DNA, transformation efficiencies of Synechocystis are very high. We have already shown that Synechocystis can be transformed with 2 plasmids. Following transformation, strains will be grown on the appropriate antibiotics and marked mutants selected. Marked mutants will then be grown on plates containing sucrose to select for unmarked mutants. Ideally we would like to introduce 4 alterations into a strain at once but even 2 alterations would double the rate of mutant production in Synechocystis.
After establishing the maximum number of plasmids that can be introduced into Synechocystis, a second set of plasmids containing the various flanking regions but with the same expression cassette, will be constructed. This expression cassette encodes the required genes for heptadecane (a component of diesel) biosynthesis and has been validated in our lab. By introducing this cassette into different regions of the genome simultaneously we hope to increase biofuel output. Heptadecane amounts will be quantified by GCMS using validated methods. If successful, development of this system will rapidly increase the speed at which this organism can be manipulated. The methods and plasmids may also be suitable to generate mutants in other naturally competent species.
Benefits and outcomes:
This project fits into the 'Synthesis and sharing of useful DNA parts or vectors' remit of the Synthetic Biology SRI. Our current methods and plasmids have been distributed to the other groups in Cambridge working on cyanobacteria (Alison Smith group, Plant Sciences; James Locke group, Sainsbury laboratory). We have also distributed our current methods and plasmids to other groups in the U.K. at Imperial College London, University of Edinburgh and the University of Reading, in addition to groups in Finland and New Zealand. If successful we expect the outcomes of this research to be of interest to most people working with cyanobacteria and possibly microbiologists working on other organisms. All plasmids and methods generated will be freely available upon request. We expect one publication in JOVE which will highlight our group's expertise in cyanobacterial genetics. If successful, this research will be published in an appropriate peer reviewed journal.
£1600 for publication costs. £2400 for laboratory reagents. i.e. molecular biology reagents, GCMS running costs and reagents. If laboratory reagent costs exceed £2400, a small amount may be covered by other grants.