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Team:Tuebingen/Notebook
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| - | + | ''These page holds a detailed and technical account of our research project.'' | |
| - | + | === Preparations === | |
| - | + | After determining our principal project idea we had to design our system prior to any work in the wet lab. Several steps were involved: | |
| - | -- | + | ==== Identification of plasmids ==== |
| + | |||
| + | We decided to use a shuttle vector which works in E. coli and S. cerevisiae. The advantage is that we can assemble and build our target constructs in the rapid growing E. coli. The shuttle vectors must meet the following demands: | ||
| + | * Multiple cloning site with XbaI and SpeI restriction sites | ||
| + | * Multiple cloning site with beta-galactosidase, so blue-white screening is possible | ||
| + | * Ampicillin resistance | ||
| + | * amino-acid genes for selection | ||
| + | * Integration site for integration in the yeast genome | ||
| + | |||
| + | After consultation with Prof. Jansen, our yeast expert, we decided to use the shuttle vectors pRS313, pRS315 and pRS316. | ||
| + | |||
| + | Due to several EcoRI and PstI restriction sites in the pRS plasmids (not located around the multiple cloning site), we can only use XbaI and SpeI for assembly. | ||
| + | |||
| + | ==== Identification of genes ==== | ||
| + | |||
| + | '''Receptors:''' Aiming at aquatic environment we chose to use the membrane progesterone receptor from the model organism ‘’Danio rerio’’ (zebra fish). The second organism was ‘’Salmo salar’’ (salmon) but we could not locate a homologous gene. Following a database-wide BLAST search we selected the membrane progesterone receptor from ‘’Xenopus laevis’’ (african clawed frog), another well-studied model organism, as our second receptor. | ||
| + | |||
| + | '''Signalling:''' The promoter targeted by the membrane receptors was already determined and proven working by Smith et al. (2008). We did not have the sequence used by Smith and decided to take the upstream sequence (approx. 600bp) of the fet3 gene. | ||
| + | |||
| + | The second part of the signalling system is the need of a repressor and repressor target. Additionally, knock-out strains not containing a working repressor have to be viable. | ||
| + | |||
| + | '''Reporter:''' We have access to the plasmid storage of the work group. Common reporters used in this yeast-based environment are firefly luciferase and beta-galactosidase. Both are available to us and have no legal issues to be published in the Parts Registry. | ||
| + | |||
| + | ==== Sequence analysis and primer design ==== | ||
| + | |||
| + | Next was the check of all sequences for unwanted restriction sites. | ||
Revision as of 19:38, 30 August 2012
Lab Notebook
Contents |
These page holds a detailed and technical account of our research project.
Preparations
After determining our principal project idea we had to design our system prior to any work in the wet lab. Several steps were involved:
Identification of plasmids
We decided to use a shuttle vector which works in E. coli and S. cerevisiae. The advantage is that we can assemble and build our target constructs in the rapid growing E. coli. The shuttle vectors must meet the following demands:
- Multiple cloning site with XbaI and SpeI restriction sites
- Multiple cloning site with beta-galactosidase, so blue-white screening is possible
- Ampicillin resistance
- amino-acid genes for selection
- Integration site for integration in the yeast genome
After consultation with Prof. Jansen, our yeast expert, we decided to use the shuttle vectors pRS313, pRS315 and pRS316.
Due to several EcoRI and PstI restriction sites in the pRS plasmids (not located around the multiple cloning site), we can only use XbaI and SpeI for assembly.
Identification of genes
Receptors: Aiming at aquatic environment we chose to use the membrane progesterone receptor from the model organism ‘’Danio rerio’’ (zebra fish). The second organism was ‘’Salmo salar’’ (salmon) but we could not locate a homologous gene. Following a database-wide BLAST search we selected the membrane progesterone receptor from ‘’Xenopus laevis’’ (african clawed frog), another well-studied model organism, as our second receptor.
Signalling: The promoter targeted by the membrane receptors was already determined and proven working by Smith et al. (2008). We did not have the sequence used by Smith and decided to take the upstream sequence (approx. 600bp) of the fet3 gene.
The second part of the signalling system is the need of a repressor and repressor target. Additionally, knock-out strains not containing a working repressor have to be viable.
Reporter: We have access to the plasmid storage of the work group. Common reporters used in this yeast-based environment are firefly luciferase and beta-galactosidase. Both are available to us and have no legal issues to be published in the Parts Registry.
Sequence analysis and primer design
Next was the check of all sequences for unwanted restriction sites.
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