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. 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.

The first thing we needed to do was to isolate the flanking sequences from Synechocystis. This was accomplished by use of 'colony PCR', the genome of Synechocystis was used as a template. We designed primers so that we would only amplify our desired sequences.

After verifying that our PCR amplified flanking sequences had the right sequence, we wanted to test them. The flanking sequences were tested by ligating them to a vector backbone, with a Kanamycin resistance insert between the two flanking sequences. The resulting plasmid had the following parts, in order:

• Left flank
• Kanamycin resistance
• Right flank
• Backbone

This plasmid was transformed into Synechocystis, and the transformed cells were cultured in BG11 growth medium containing kanamycin. Growth of Synechocystis cells in this medium, along with band shift colony PCR (See figure below), confirmed to us that the plasmid had been successfully taken up and integrated into the host genome through homologous recombination.

We wanted to see if transforming the gene glucose oxidase into Synechocystis would lead to an increased rate of carbon fixation pr. growth rate.

To achieve this we constructed a plasmid with eight parts:

• "Right flank" sequence
• Constitutionally active promoter + RBS
• LacI repressor gene
• Kanamycin resistance
• LacI inducible promoter + RBS
• Glucose Oxidase
• "Left flank" sequence
• Plasmid backbone

As growth in Synechocystis is very slow, we did not have time to test this plasmid, and so it will not be submitted as a composite BioBrick.

Instead, the plasmid was split up into several BioBricks, so that each BioBrick contains one part of the composite plasmid. The BioBricks are as follows:

• Right flank (Cloned from Synechocystis )
• Left flank (Cloned from Synechocystis )
• LacI inducible promoter + Ribosomal Binding Site (Codon optimized for Synechocystis )
• Kanamycin resistance gene (Cloned from Synechocystis )
• Glucose Oxidase (Synthesized by GenScript)
• Right flank + Kanamycin resistance + Right flank

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

Future efforts

Our candiate gene for increasing CO₂ the fixation rate 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.

Future efforts should focus on transforming the composite plasmid containing all 6 BioBricks into Synechocystis, and testing the carbon fixation rate of the resulting transformants. Such testing requires specialized equipment that is able to measure minute differences in the partial pressure of CO<sub>2.

We had plans of collaborating with SINTEF CO2-capture and storage, which were kind enough to offer us the use of their facilities and equipment. Doing our planned experiments in their facilities would have provided us with evidence for the effect of glucose oxidase on Synechocystis' carbon fixation rate.</sub>