Part:BBa_K2148002:Experience

The aphA6 gene was assembled into a level 1 alpha plasmid with two other parts from our library: homology region with psaA 5' UTR and promoter and the 3' UTR rbcL with homology region. This generated a plasmid which could be used to transform Chlamydomonas reinhardtii chloroplasts by insertion through homologous recombination. We shot this construct into cultures of the CW15 C. reinhardtii biolistically.

The cultures grew (as shown below in image) on kanamycin+TAP plates, showing the functionality of this part. Colonies were indeed seen to have grown on the negative control plates but we believe this to be due to our own contamination in the procedure because:

(a) This antibiotic resistance is not used in the Plant Sciences Dept of Cambridge (where the team is based)

(b) Could possible be the case that a layer of cells has died (due to lack of resistance) and the new colonies are forming upon these, avoiding contact with the antibiotic. However, we replated the colonies onto new antibiotic+TAP plates and the cells were able to grow well.

PCR analysis was not conclusive on the result of the biolistic transformation. Ideally, would need to repeat the experiment (time constraints did not allow us to).

INCLUDE GEOFF'S PHOTOS OF COLONY

Applications of BBa_K2148002

Used as a selection pressure for retention of gene of interest and achievement of homoplasmy when transforming Chlamydomonas reinhardtii chloroplasts.

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