Template:Team:Bonn:NetworkData

content.childs=[35];

content.childs=[35];

content.titleShort = "pDawn";

content.titleShort = "pDawn";

content.summary= "The plasmid pDawn was designed by Ohlendorf et al.^{<a href=#ref21.1>21.1</a>} in 2012 together with its counter plasmid pDusk. Both plasmids are single plasmid systems, which allow the activation (pDawn) or repression (pDusk) of gene expression by blue light. They are easy to implement in the laboratory and lead to up to 460-fold activity change upon ilumination.";

content.summary= "The plasmid pDawn was designed by Ohlendorf et al.^{<a href=#ref21.1>21.1</a>} in 2012 together with its counter plasmid pDusk. Both plasmids are single plasmid systems, which allow the activation (pDawn) or repression (pDusk) of gene expression by blue light. They are easy to implement in the laboratory and lead to up to 460-fold activity change upon ilumination.";

content.text= "The plasmid pDawn was designed by Ohlendorf et al.^{<a href=#ref21.1>21.1</a>} in 2012 together with its counter plasmid pDusk. Both plasmids are single plasmid systems, which allow the activation (pDawn) or repression (pDusk) of gene expression by blue light. They described their system as easy to implement in the laboratory and suitable for both preparative and analytical purposes. Further, they were able to show up to 460-fold induction upon ilumination using their system. </br></br> Because, pDawn (and pDusk) is a system that regulates gene expression and thus acts on DNA level it is an eligible candidate for us to compare the speed of action of our system, which acts on protein level to an DNA regulation system. As an additional benefit, with pDawn, we implement a powerful light-induceable system, with low background expression and high dark/light expression difference in the iGEM database. </br></br> Ohlendorf et al. (2012)^{<a href=#ref21.1>21.1</a>} had previously designed the histidine kinase YF1, which phosphorylates FixJ in the absence of blue light. The phosphorylated FixJ drives robust gene expression from the FixK2 promotor as shown in figure 1 for both pDawn and the pDusk. Upon light absorption, net kinase activity of YF1 and consequently gene expression under control of the FixK2 promotor is greatly reduced. pDusk an pDawn enable light-repressed or light-induced gene expression in Escherichia coli and are easy to implement in the laboratory. Therefore, all components of the YF1/FixJ TCS were assembled on only one medium-copy plasmid in which YF1 and FixJ are constitutively expressed by the Lac^q promotor. Target genes can be introduced via the multiple-cloning site (MCS) under the control of the pFixK2 promotor, allowing light-repressed gene expression. To obtain pDawn the light effect is inverted by the introduction of a gene-inversion cassette into pDusk. This gene-inversion cassette is based on the &lambda; phage repressor cI and the &lambda; phage promotor pR. (see figure 1)</br></br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/a/a1/Bonn_MS_Figure1_pDusk%26pDawn.jpg></br>Figure 1: Plasmids pDusk an pDawn. YF1 and FixJ are constitutively expressed by the Lacq promotor. Target genes can be introduced via the multiple-cloning site (MCS) under the control of the pFixK2 promotor. (Ohlendorf et al. 2012^{<a href=#ref21.1>21.1</a>})</div></br>Ohlendorf et al. (2012)^{<a href=#ref21.1>21.1</a>} analyzed the system by the insertion of the red-fluorescent reporter protein DsRed Express2. That way they were able to measure up to 460-fold induction upon ilumination. However, they also found that their plasmid, which acts on gene expression level, only allows light-induced perturbations on the timescale of hours.</br></br><h2>References</h2></br><p><a name=ref21.1>21.1</a> <a href=http://www.sciencedirect.com/science/article/pii/S0022283612000113> Ohlendorf et al. (2009) From Dusk till Dawn One-Plasmid Systems for Light-Regulated Gene Expression.</a></p>";   

content.text= "The plasmid pDawn was designed by Ohlendorf et al.^{<a href=#ref21.1>21.1</a>} in 2012 together with its counter plasmid pDusk. Both plasmids are single plasmid systems, which allow the activation (pDawn) or repression (pDusk) of gene expression by blue light. They described their system as easy to implement in the laboratory and suitable for both preparative and analytical purposes. Further, they were able to show up to 460-fold induction upon ilumination using their system. </br></br> Because, pDawn (and pDusk) is a system that regulates gene expression and thus acts on DNA level it is an eligible candidate for us to compare the speed of action of our system, which acts on protein level to an DNA regulation system. As an additional benefit, with pDawn, we implement a powerful light-induceable system, with low background expression and high dark/light expression difference in the iGEM database. </br></br> Ohlendorf et al. (2012)^{<a href=#ref21.1>21.1</a>} had previously designed the histidine kinase YF1, which phosphorylates FixJ in the absence of blue light. The phosphorylated FixJ drives robust gene expression from the FixK2 promotor as shown in figure 1 for both pDawn and the pDusk. Upon light absorption, net kinase activity of YF1 and consequently gene expression under control of the FixK2 promotor is greatly reduced. pDusk an pDawn enable light-repressed or light-induced gene expression in Escherichia coli and are easy to implement in the laboratory. Therefore, all components of the YF1/FixJ TCS were assembled on only one medium-copy plasmid in which YF1 and FixJ are constitutively expressed by the Lac^q promotor. Target genes can be introduced via the multiple-cloning site (MCS) under the control of the pFixK2 promotor, allowing light-repressed gene expression. To obtain pDawn the light effect is inverted by the introduction of a gene-inversion cassette into pDusk. This gene-inversion cassette is based on the &lambda; phage repressor cI and the &lambda; phage promotor pR. (see figure 1)</br></br><div class=contant-image><img src=https://static.igem.org/mediawiki/2013/a/a1/Bonn_MS_Figure1_pDusk%26pDawn.jpg></br>Figure 1: Plasmids pDusk an pDawn. YF1 and FixJ are constitutively expressed by the Lacq promotor. Target genes can be introduced via the multiple-cloning site (MCS) under the control of the pFixK2 promotor. (Ohlendorf et al. 2012^{<a href=#ref21.1>21.1</a>})</div></br>Ohlendorf et al. (2012)^{<a href=#ref21.1>21.1</a>} analyzed the system by the insertion of the red-fluorescent reporter protein DsRed Express2. That way they were able to measure up to 460-fold induction upon ilumination. However, they also found that their plasmid, which acts on gene expression level, only allows light-induced perturbations on the timescale of hours.</br></br><h2>References</h2></br><p><a name=ref21.1>21.1</a> <a href=http://www.sciencedirect.com/science/article/pii/S0022283612000113> Ohlendorf et al. (2009) From Dusk till Dawn One-Plasmid Systems for Light-Regulated Gene Expression.</a></p>";