Difference between revisions of "Part:BBa K1465201"
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| + | ===Characterization=== | ||
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| + | <div class="element" style="margin:10px; padding:10px; text-align:center; width:300px;float:right"> | ||
| + | <a href="https://static.igem.org/mediawiki/2014/a/a8/Bielefeld-CeBiTec_2014-10-11_prkA-toxicity.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/a/a8/Bielefeld-CeBiTec_2014-10-11_prkA-toxicity.png" width="300px"></a><br> | ||
| + | <font size="2" style="text-align:center;"><b>Figure 1</b>: Toxicity of phosphoribulokinase without RuBisCO</font> | ||
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| + | The sequence of the phosphoribulokinase was synthesized to remove illegal restriction sites and to optimize the codon usage for <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a>. We were able to transform the prkA (<a href="https://parts.igem.org/Part:BBa_K1465201" target="_blank">BBa_K1465201</a>, <a href="https://parts.igem.org/Part:BBa_K1465212" target="_blank">BBa_K1465212</a>, <a href="https://parts.igem.org/Part:BBa_K1465214" target="_blank">BBa_K1465214</a>) into <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> but without ribosom binding site. As a template for the synthesis we used the prkA of <i>Synechococcus elongatus</i>.<br> | ||
| + | The toxicity of the PrkA in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> was described previously by Parikh et al., 2006 and Bonacci et al., 2012. The toxicity results through the accumulation of ribulose 1,5-bisphosphate which can not be further metabolized as shown in Figure 1. | ||
| + | We performed an enzyme assay to identify the functionality of the PrkA in <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a>. In order to investigate this we cultivated the strain and made a crude cell extract. The cell extract was incubated for 1 h at 37°C. We compared different approaches. First we incubated a wild type strain, secondly we incubated the crude cell extract and thirdly we incubated the crude cell extract with 1 mM ribulose 5-phosphate which is the substrate for the PrkA. The aim was to identify ribulose 1,5-bisphosphate with the HPLC. Because the wild type is not able to produce ribulose 1,5-bisphosphate the PrkA activity should be easily observed. We were not able to identify the product, ribulose 1,5-bisphosphate, with HPLC in all approaches. | ||
| + | To perform a SDS-Page we cultivated <i>E. coli</i> carrying prkA strain and induced gene expression with 1 mM IPTG (<a href="https://parts.igem.org/Part:BBa_K1465212" target="_blank">BBa_K1465212</a>, <a href="https://parts.igem.org/Part:BBa_K1465214" target="_blank">BBa_K1465214</a>). In comparison to the wild type the prkA carrying strain showed similar growth behavior. The resulting SDS-Page is shown below.<br> | ||
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| + | <a href="https://static.igem.org/mediawiki/2014/e/e0/Bielefeld-CeBiTec_14-10-16_ptac_prkA.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2014/e/e0/Bielefeld-CeBiTec_14-10-16_ptac_prkA.jpg" width="350px"></a><br> | ||
| + | <font size="2" style="text-align:center;"><b>Figure 2</b>: SDS-Page of ptac prkA</font> | ||
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| + | The PrkA has a molecular size of around 38 kD. There is a clear band shown between 35 kD and 40 kD. This fragment was cut out to identify the protein via MALDI-TOF analysis. With the MALDI-TOF we were able to identify three peptides of the PrkA which is sufficient.<br> | ||
| + | The results of our overall approaches looked like the PrkA is expressed by <a href="http://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli" target="_blank"><i>E. coli</i></a> but does not show functionality in an <i>in vitro</i> assay. A possible problem is the light dependent activation of the PrkA. The light triggers the PSI in photosynthetic active organisms. This trigger reduces ferredoxine. In the follwing reaction thioredoxin is reduced while ferredoxin is oxidized again. The reduced thioredoxin is able to break disulfides. The PrkA activation is triggered by thioredoxin which is maybe not present sufficiently in <i>E. coli</i>. As previously described it would be possible to activate the PrkA by adding DTT in the enzyme assay (Hariharan et al., 1998) which would be our next target for an enzyme assay. | ||
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Revision as of 03:47, 18 October 2014
Phosphoribulokinase prkA from Synechococcus elongatus
Phosphoribulokinase prkA from Synechoccus elongatus, codon optimized for E. coli.
Usage and Biology
The phosphoribulokinase is the enzyme which catalyzes the irreversible reaction from ribulose 5-phosphate to ribulose 1,5-bisphosphate under consumption of ATP. The phosphoribulokinase is an enzyme unique to the Calvin cycle (Rumpho et al., 2009). All photosynthetic organisms depend on the PrkA to sustain their cyclic activity. Several information like regulation and protein activity are well characterized for plants and also for cyanobacteria (Wadano et al., 1995). In many organisms the PrkA is activated in the presence of light which is thioredoxin mediated (Reduction of intramolecular disulfides). DTT may stimulate the enzyme activity in certain cases (Hariharan et al., 1998) which could be a useful information if the expression of the prkA is not strong enough in E. coli.
Figure 3: Reaction of phosphoribulokinase
Characterization
Figure 1: Toxicity of phosphoribulokinase without RuBisCO
The toxicity of the PrkA in E. coli was described previously by Parikh et al., 2006 and Bonacci et al., 2012. The toxicity results through the accumulation of ribulose 1,5-bisphosphate which can not be further metabolized as shown in Figure 1. We performed an enzyme assay to identify the functionality of the PrkA in E. coli. In order to investigate this we cultivated the strain and made a crude cell extract. The cell extract was incubated for 1 h at 37°C. We compared different approaches. First we incubated a wild type strain, secondly we incubated the crude cell extract and thirdly we incubated the crude cell extract with 1 mM ribulose 5-phosphate which is the substrate for the PrkA. The aim was to identify ribulose 1,5-bisphosphate with the HPLC. Because the wild type is not able to produce ribulose 1,5-bisphosphate the PrkA activity should be easily observed. We were not able to identify the product, ribulose 1,5-bisphosphate, with HPLC in all approaches. To perform a SDS-Page we cultivated E. coli carrying prkA strain and induced gene expression with 1 mM IPTG (BBa_K1465212, BBa_K1465214). In comparison to the wild type the prkA carrying strain showed similar growth behavior. The resulting SDS-Page is shown below.
Figure 2: SDS-Page of ptac prkA
The results of our overall approaches looked like the PrkA is expressed by E. coli but does not show functionality in an in vitro assay. A possible problem is the light dependent activation of the PrkA. The light triggers the PSI in photosynthetic active organisms. This trigger reduces ferredoxine. In the follwing reaction thioredoxin is reduced while ferredoxin is oxidized again. The reduced thioredoxin is able to break disulfides. The PrkA activation is triggered by thioredoxin which is maybe not present sufficiently in E. coli. As previously described it would be possible to activate the PrkA by adding DTT in the enzyme assay (Hariharan et al., 1998) which would be our next target for an enzyme assay.