For easier understanding the BioBricks for our project may be sorted into four groups: (1) the creation of a vector in which you may clone every protein of interest, which gets then linearized and becomes used for transformation of Physcomitrella patens, (2) BioBricks which can be used to determine the localization of a protein in Physcomitrella patens being either cytoplasmatic, secreted or receptor bound, (3) a set of six different phytoremediation effector proteins which either bind (BioAccumulation) or degrade (BioDegradation) a xenobiotic substance and finally (4) we assembled the BioBricks for a red light triggered kill switch that kills transgenic moss cells if they are exposed to full spectrum sun light.

Figure 1: Overview over the BioBricks used in TU-Munich 2013 iGEM project.

Best New BioBrick Part candidates

  • Natural: BBa_K1159000: Erythromycin Esterase Type II (EreB) in RFC[25]

BioBricks we improved

Conversion to RFC 25

Some BioBricks which were already present in the parts registry were converted from RFC 10 to RFC 25 by PCR. The purpose of this improvement was that we introduced with our project the possiblility to dictate the cellular localisation of proteins (cytosolic, secreted and receptor bound). For this purpose the open reading frame of the protein has to be fused to a N-terminal signal peptide (for secretion) or has to be inserted into the extracellular domain of a synthetic receptor. We converted the following BioBricks to RFC 25:

  • Laccase BBa_K863000 was improved to the BioBrick BBa_K1159002
  • Protein phosphatase 1 is a BioBrick BBa_K1012001 created by 2013 Dundee iGEM team. We have also thought to target microcystin as an important environmental toxin. We liked the idea of using the protein affected in the natural toxicity mechanism to solve the problem. Therefore we contacted Dundee team, they sent us their BioBrick which we converted to RFC 25. This was necessary as we wanted to localise it extracellularely in a receptor of our moss.

Adding improved versions of the protein

The fluoresceine binding Anticalin FluA was already present in the Parts Registry in RFC 25. As this BioBrick is from 2008 we used a higher engineered variant with three additional mutations which make it 75-times more affine to fluorescein (KD from 152 nM to 2 nM). This seemed an imporatant point as this Anticalin was twice present in the finals of iGEM (Freiburg Team 2008 and 2009) and an improved variant may be helpful for further iGEM teams as well as for our purpose to use it as a binding protein on trangenic moss. The outdated FluA with a lower affinity was BBa_K157004 and the newer version is now available as BBa_K1159003.

Correcting the incorrect sequence of a working BioBrick

When we designed our synthetic receptor for Physcomitrella patens we planed to fuse a fluorescent protein to the cytoplasmatic domain of this receptor in order to be able to localize it by fluorescence microscopy. For this purpose we needed a fluorescent protein in RFC 25 which can be used to create the desired protein fusion. This BioBricks we obtained from the LMU iGEM Team 2012 who had created this BioBrick. After fetching it and a initial sequecing we realised that there was a false sequence in the Parts Registry and that it is not GFPmut3 but GFPmut1 which they provided us with. We finally used this fluorescent protein for our project and put the correct seqeuence into the Parts Registry (see BBa_K1159311).

Impressions from the packaging of our BioBricks

Based on the registry reforms Andreas packed one box containing our 72 Biobricks which were send on the 19 th of September.

Figure 2: Successful preparing of the BioBrick sendbox
Figure 3: One of our BioBrick sendboxes