Team:TU-Munich/Results/BioBricks

From 2013.igem.org

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Revision as of 22:18, 2 October 2013

Sendbox

Igkappa-GFP

<groupparts>iGEM2013 TU-Munich</groupparts>

Figure 1:Successful preparing of the biobrick sendbox

Figure 2:One of our biobrick sendboxes

BioBricks we improved

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 <partinfo>BBa_K863000</partinfo> was improved to the BioBrick <partinfo>BBa_K1159002</partinfo>
Protein phosphatase 1 is a BioBrick 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.

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 (K_D 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 The outdated FluA with a lower affinity was <partinfo>BBa_K157004</partinfo> and the newer version is now available as <partinfo>BBa_K1159002</partinfo>.

BioBricks we characterized

LMU-GFP

References:

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984

http://www.ncbi.nlm.nih.gov/pubmed/6327079 Edens et al., 1984 Edens, L., Bom, I., Ledeboer, A. M., Maat, J., Toonen, M. Y., Visser, C., and Verrips, C. T. (1984). Synthesis and processing of the plant protein thaumatin in yeast. Cell, 37(2):629–33.

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 == BioBricks we improved ==
-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]:
+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 <partinfo>BBa_K863000</partinfo> was improved to the BioBrick <partinfo>BBa_K1159002</partinfo>
 *Protein phosphatase 1 is a BioBrick 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.
-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 (K<sub>D</sub> 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 <partinfo>BBa_K157004</partinfo> and the newer version is now available as <partinfo>BBa_K1159002</partinfo>.
+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 (K<sub>D</sub> 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 <partinfo>BBa_K157004</partinfo> and the newer version is now available as <partinfo>BBa_K1159002</partinfo>.
 == BioBricks we characterized ==