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Team:Heidelberg/Templates/Indigoidine week5 overview
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| - | At the beginning of our wetlab phase, we wanted to transform ''E. coli'' cells with plasmids containing an | + | At the beginning of our wetlab phase, we wanted to transform ''E. coli'' cells with plasmids containing an indigoidine synthetase and a 4'-Phosphopanthetheinyl-transferase (PPTase) to see whether we can observe blue colonies on our plates as this has been reported by groups working with indigoidine synthetases (Takahashi 2007, Brachmann2012). <br/> |
| - | We focused on the work of Marius Müller et. al. in 2012 ( | + | We focused on the work of Marius Müller et. al. in 2012 (Muller 2012). The group used the bpsA indigoidine synthetase (''blue pigment synthetase A'') from ''S. lavendulae'' ATCC11924 (Takahashi 2007) and the PPTase svp from ''S. verticillus'' ATCC15003 (Sanchez 2001) to establish both a fluorescence and a chromophore based reporter assay for mammalian cells by expression of bpsA and svp which results in production of a blue pigment/ fluorophore. The group kindly supported us by sending two of their constructs, namely the pET derived expression vectors pMM64 carrying the bpsA indigoidine synthetase with an ampicillin resistance gene and pMM65 carrying svp and a kanamycin resistance gene. Both bpsA and svp rom the Fussenegger lab are codon-optimized versions for expression in eukaryotic cell lines. <br/> |
| - | We transformed competent | + | We transformed competent ''E. coli'' TOP10 with each plasmid to prepare plasmid DNA and perform a cotransformation of an ''E. coli'' Rosetta strain with both plasmids. Transformed cells which carry both plasmids should produce the blue pigment indigoidine. |
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| - | Unfortunately, there were no blue colonies after the co-transformation. We repeated the experiment under various growth conditions; i.e. we used different incubation temperatures, light conditions, shaking and addition of ascorbic acid, which was reported to stabilize indigoidine in liquid cultures ( | + | Unfortunately, there were no blue colonies after the co-transformation. We repeated the experiment under various growth conditions; i.e. we used different incubation temperatures, light conditions, shaking and addition of ascorbic acid, which was reported to stabilize indigoidine in liquid cultures (Muller 2012). |
</p> | </p> | ||
Latest revision as of 14:50, 21 October 2013
Indigoidine Production - bpsA
At the beginning of our wetlab phase, we wanted to transform E. coli cells with plasmids containing an indigoidine synthetase and a 4'-Phosphopanthetheinyl-transferase (PPTase) to see whether we can observe blue colonies on our plates as this has been reported by groups working with indigoidine synthetases (Takahashi 2007, Brachmann2012).
We focused on the work of Marius Müller et. al. in 2012 (Muller 2012). The group used the bpsA indigoidine synthetase (blue pigment synthetase A) from S. lavendulae ATCC11924 (Takahashi 2007) and the PPTase svp from S. verticillus ATCC15003 (Sanchez 2001) to establish both a fluorescence and a chromophore based reporter assay for mammalian cells by expression of bpsA and svp which results in production of a blue pigment/ fluorophore. The group kindly supported us by sending two of their constructs, namely the pET derived expression vectors pMM64 carrying the bpsA indigoidine synthetase with an ampicillin resistance gene and pMM65 carrying svp and a kanamycin resistance gene. Both bpsA and svp rom the Fussenegger lab are codon-optimized versions for expression in eukaryotic cell lines.
We transformed competent E. coli TOP10 with each plasmid to prepare plasmid DNA and perform a cotransformation of an E. coli Rosetta strain with both plasmids. Transformed cells which carry both plasmids should produce the blue pigment indigoidine.
Unfortunately, there were no blue colonies after the co-transformation. We repeated the experiment under various growth conditions; i.e. we used different incubation temperatures, light conditions, shaking and addition of ascorbic acid, which was reported to stabilize indigoidine in liquid cultures (Muller 2012).
