Part:BBa_K5023000:Experience

In our research with Chlamydomonas reinhardtii, a green alga, we've consistently utilized the bleomycin resistance gene, commonly known as the "ble" gene. This gene serves as a selectable marker, enabling us to easily identify cells that have successfully integrated our desired genetic modifications.

Here's how we proceed:

Preparation: Initially, we incorporate the ble gene into a plasmid. Alongside the ble gene, we also insert the specific gene of interest (Like Fast-PETase) that we aim to introduce into the Chlamydomonas cells.

Transformation: With our vector prepared, we introduce it into Chlamydomonas reinhardtii cells. Depending on the specific requirements of our experiment, we employ methods like electroporation or glass bead agitation for this purpose.

Selection: After the transformation process, we expose the Chlamydomonas cells to zeocin, an antibiotic that belongs to the bleomycin family. The ble gene confers resistance to zeocin. As a result, only the cells that have successfully integrated and expressed the ble gene can survive in the presence of this antibiotic.

Analysis: After allowing some time for the cells to grow, we analyze the surviving colonies to confirm the presence and expression of our gene of interest. The ble gene acts as a marker, signaling which cells have successfully taken up the vector.

By using the ble gene as a selectable marker, we've been able to streamline our work with Chlamydomonas reinhardtii. It provides us with a reliable and efficient method to identify transformed cells, enhancing the precision of our genetic modification experiments.

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