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Team:Valencia/genetic engineering
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Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO2 and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br> | Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO2 and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria <i>Synechococcus elongatus</i> for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).<br><br> | ||
S. elongatus has a circular genome of ≈2.7Mb (fully sequenced link a la base de datos) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). | S. elongatus has a circular genome of ≈2.7Mb (fully sequenced link a la base de datos) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009). | ||
| - | ¿¿Nuestro objetivo y constructo final?? | + | ¿¿Nuestro objetivo y constructo final??<br><br><br><br> |
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| + | <strong>Transforming Synechococcus elongatus PCC7942 for promoter characterization:</strong> | ||
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| + | Our main objective has been to characterize the psbAI promoter (link a submitted parts) of Synechococcus elongatus PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (link a prototipo) to have a diel switch of AHL, the signal molecule for our Aliivibrio fischeri population to glow. | ||
| + | To characterize this promoter we had two options: making psbAIp: lacZ fusions and monitored the β-galactosidase activity or making psbAIp: luxABCDE fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a psbAIp::lacZ fusion because some assays report that the psbAI promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choose using psbAIp:luxABCDE vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in E. coli and transformed them into S. elongatus, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab. | ||
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Revision as of 16:33, 26 September 2012
Genetic Engineering
¿¿Nuestro objetivo y constructo final??
Cyanobacteria are great organisms to be used in Synthetic Biology because of their ability to capture solar energy and CO2 and the fact that they can be easily genetically manipulated due to its small genome and their capacity to accept foreign DNA naturally. For our project we have chosen the cyanobacteria Synechococcus elongatus for genetic engineering because is the model organism for studying some prokaryotic processes (there is a lot of information of how to transform it) and in the last years it has become a model organism for some industrial processes, like biofuel production (Wang et al. 2012).
S. elongatus has a circular genome of ≈2.7Mb (fully sequenced link a la base de datos) with a GC content of 55.5%, which contains the genes for 2.612 proteins and 53 RNAs (Atsumi et al. 2009).
¿¿Nuestro objetivo y constructo final??
Transforming Synechococcus elongatus PCC7942 for promoter characterization:
Our main objective has been to characterize the psbAI promoter (link a submitted parts) of Synechococcus elongatus PCC7942 in order to know more about its operation and understand how this promoter would control our final construct (link a prototipo) to have a diel switch of AHL, the signal molecule for our Aliivibrio fischeri population to glow.
To characterize this promoter we had two options: making psbAIp: lacZ fusions and monitored the β-galactosidase activity or making psbAIp: luxABCDE fusions and monitored the bioluminescence produced as a result of the promoter activation. We refused to use a psbAIp::lacZ fusion because some assays report that the psbAI promoter response to light cannot be properly monitored by the β-galactosidase activity (Nair et al. 2001). For this reason we choose using psbAIp:luxABCDE vector fusions. With this aim, we cloned several fusion plasmids (pAM977 and pAM2195) in E. coli and transformed them into S. elongatus, both wildtype and cscB strain. The fusion vectors were provided by Susan Golden´s lab.
