iGEM 2023 — Team patrasbio


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## 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.

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## Results

The transformation of bacterial strains was successful, resulting in cells carrying tachyplesin. However, due to limited time and resources within the scope of the iGEM competition, we were unable to conduct the necessary assessment of tachyplesin's antimicrobial activity against microorganisms colonizing and causing biodeterioration in historic marble monuments, despite this being the primary aim of our project, M.ER.CURE 2.0. This highlights the importance of ongoing research and development in this field to achieve sustainable solutions for the protection of cultural heritage from biodegradation.

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