Team:Macquarie Australia/Project/background

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Photosynthesis + Photosystem 2

Mutagenesis

Mutagenesis techniques have been an important tool for scientists to determine the individual functions of the genes at each step shown in Figure 1. For example, Meinecke et al conducted a study on Chlamydomonas reinhardtii that were chlorophyll deficient. These mutants were found to have a build up of Mg-protoporphyrin IX as a result of mutations in the chlM gene. The chlM gene encodes the enzyme Mg-protoporhyrin IX methyltransferase and this buildup of substrate resulted in no production of chlorophyll, suggesting that the chlM gene was an essential step in the chlorophyll biosynthetic pathway (6).
Whilst it is known how the chlorophyll biosynthetic pathway operates, currently there has been little success in replicating this system synthetically. Hence, this serves as the basis for this research project. If the chlorophyll biosynthetic pathway is successfully created, it will be a world first advancement in this area.

Chlorophyll

Chlorophyll is a photosynthetic pigment differentiated from other tetrapyrroles by the presence of a magnesium ion in its structure (3). Currently, four types of chlorophyll pigments have been identified including chlorophyll a, b, c and d. Recently a fifth type, chlorophyll f, has been discovered as an accessory pigment in a cyanobacterium (4). Before the discovery that the cyanobacterium Acaryochloris marina used chlorophyll d as its primary photosynthetic pigment, it was thought that only chlorophyll a was utilised by the photosynthetic reaction centres of all photosynthetic organisms (5). In this study we are interested in chlorophyll a, as this is one of the main photosynthetic pigments produced in Chlmaydomonas reinhardtii, the organism from which the genes necessary for out project have been sourced(6).

Gibson Cloning

Gibson cloning is a powerful and innovative method of DNA polymer synthesis, devised by DG Gibson et al. in 2009 (Gibson et al., 2009). The technique comprises an isothermal, single-reaction experiment involving the concerted action of a 5’-exonuclease, a DNA polymerase and a DNA ligase. An overview of the Gibson assembly method is provided in Figure 1 below and involves the ligation of DNA oligos, or ‘gBlocks’, of varying size (upto 500 BP each). As can be seen in the figure, gBlocks contain overlapping DNA regions of at least 30 BP which are exposed upon the action of the exonuclease. This facilitates complimentary base pairing between gBlocks. The simultaneous action of a DNA polymerase (preferably with proof-reading activity) and Taq polymerase, respectively, then complete and seal the newly formed conjugate (Gibson et al., 2010, Gibson et al., 2009).

The technique has obvious advantages in terms of time efficiency and simplicity. It involves considerably less labour and steps to complete compared to traditional methods. In particular there are no polymerase cycling assembly; PCR, gel purification; restriction digestion and DNA ligation steps are necessary (Gibson et al., 2010). For these reasons we have chosen to apply this technique to the assembly our BioBricks.

Figure 1: An overview of the key steps in the Gibson assembly method for DNA polymer synthesis. (Figure adapted from DG Gibson et al.)