Engineering Success

Design:

We designed 2 types of parts: Parts which encode Tachyplesin, so they have the sequence of the Tachyplesin gene and parts which do not encode Tachyplesin and they have the same sequence with the first half ones, except for the Tachyplesin gene, which is missing. These would be used as controls. Before the Tachyplesin gene (where existing) we put the gene of sfGFP (fluorescent) , so that would be able to detect the expressed protein with blue light.

Build:

All parts except for the basic Tachyplesin, I11S and Y8S parts, begin with the T7 promoter, which is being activated by T7 RNA polymerase. Our experiments included the transformation of DH5A E.Coli cells with the parts and the last stage was the isolation of the expressed protein.

Test:

We transformed the DH5A cells with the ligation products and realized with blue light that there was not GFP, so we understood that the protein was not expressed. At first we were very confused, but we realized that DH5A do not have T7 RNA polymerase, so the T7 promoter was not activated in order to express the protein.

Learn:

We learned that DH5A are not the appropriate cells for expressing proteins, but they are useful just for cloning. Then, we read that we have to use BL-21 E.Coli cells, which have T7 RNA polymerase and thus are able to express proteins.