Introduction

CO₂ emissions have recieved a lot of attention in modern times, due to concerns that high emission levels are facilitating global warming. Consequently, a lot of research is focused on ways of reducing CO₂ emissions from industry, and ways of fixating atmospheric CO₂ at a greater than normal rate.

Our project is attempting to produce a plasmid, which when placed inside photosynthetic bacteria, increases their rate of CO₂ fixation. In order to achieve this, we first need to construct BioBricks that allow inducible expression of non native genes in our chassis; Synechocystis sp. PCC 6803, when assembled.

Making a Synechocystis compatible vector

In order to start working with Synechocystis sp. PCC 6803, we have to construct a plasmid that will allow insertion of foreign genes into the Synechocystis genome. As Synechocystis does not retain inserted plasmids, transformations must make use of conjugation in order to make the insert part its genome.

To achieve this we have constructed a plasmid with eight parts:

• "Right flank" sequence
• Constitutionally active promoter + RBS
• LacI repressor gene
• Kanamycin resistance
• LacI inducible promoter + RBS
• Red fluorescent protein
• "Left flank" sequence
• Plasmid backbone

The flank sequences have been chosen such that they are homologous to a pair of sequences in a neutral site of the Synechocystis genome. When this plasmid is transformed into Synechocystis, the DNA between the two flank sequences is conjugated into its genome. By growing cultures in a selection medium containing Kanamycin, the insert is kept inside the genome, allowing us to express foreign genes inside the organism.

Since we are submitting to the iGEM registry, the plasmid will be split up into several BioBricks which each contain one of the parts of the complete plasmid. The BioBricks are as follows:

• Right flank (Cloned from Synechocystis )
• Left flank (Cloned from Synechocystis )
• LacI inducible promoter + RBS (Codon optimized for Synechocystis )
• Kanamycin resistance gene (Cloned from Synechocystis )

These BioBricks should allow teams the means to use Synechocystis as a chassis in future iGEM competitions.

Increasing the CO₂ fixation rate

After verifying that the expression system is working as intended in Synechocystis, we will replace the fluorescent protein gene with one that is predicted to increase the rate of carbon fixation in the organism. The way we will idenfity this gene is by use of metabolic modelling. Once a candidate gene has been detected, it will be cloned and transformed into Synechocystis.

One candiate gene is glucose oxidase, which is not originally present in Synechocystis. This gene encodes the enzyme Glucose Oxidase, which essentially reduces the oxygen concentration inside the cell. RuBisCO, the CO₂ fixating enzyme in photosynthetic organisms, has a high affinity for binding O₂, which can interfere with CO₂ binding. Reducing O₂ concentrations could therefore lead to an increased rate of CO₂ fixation in Synechocystis.