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Anti-freeze protein

The antifreeze protein used in this project is normally produced by an insect, the Spruce Budworm, Choristoneura fumiferana[5]. The structure of the antifreeze protein is composed of beta-sheets stacked parallely. From a cross section the protein appears triangular with rectangular sides[6] (Fig. 1).

Figure 1: Protein structure models of the antifreeze protein from the spruce budworm. Structures were determined with X-ray diffraction, 2.30 Å [12]

Through uniprot we found the aminosequence of the protein, where it had the entry name Q9GTP05,7⁠. We then converted the sequence of amino acids into DNA codons. This DNA sequence were then codon optimized for Physcomitrella patens. Lastly, we sent the DNA sequence to IDT, where the gene was synthesized. The aim is to confirm expression of this novel gene in P. Patens and add it as a new biobrick to the registry.

Resveratrol

Resveratrol is an antioxidant. Resveratrol is synthesized from p-Coumaroyl-CoA by stilbene synthase (STS) making the final enzyme in the resveratrol biosynthesis. Moss already produces the precursor p-Coumaroyl-CoA, which makes it simple to engineer moss to produce resveratrol.

Physcomitrella patens has been shown to produce enzymes similar to 4CL. These enzymes from the Pp4CL family (Physcomitrella patens) have been shown to have similar function as enzymes from the 4CL family of higher plants [9]. This means that P. Patens only lack the production of STS to produce resveratrol.

Since the STS is already in the registry (Part:BBa_K1033002) but is not available, we secured the STS-gene from our department (PLEN, Plant and Environmental sciences, thanks to Brian King). The aim is to improve the biobrick (Bba_K1033002) by expressing STS in P. Patens and detect resveratrol using Liquid chromatography–mass spectrometry (LCMS).

Constructs

The construct for introduction and expression of the anto-freeze protein or STS contains are flanked by regions homologous to the moss genome so that the STS-gene will integrate by homologous recombination in moss. The constructs furthermore contains the ZmUbi-promoter, a kanamycin resistance gene and YFP, to confirm transformation. Three pieces are amplified by PCR and assembled by homologous recombination in moss. Click on the construct to learn more about the different parts. (Works only for the lower construct so far...).