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| | {{:Team:Tuebingen/Template/Tuebingen}} | | {{:Team:Tuebingen/Template/Tuebingen}} |
| | + | = Lab Notebook = |
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| - | == Lab Notebook ==
| + | ''The following pages hold a detailed and technical account of the developement of our research project.'' |
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| - | __TOC__
| + | * [[Team:Tuebingen/NotebookPreparations|Preparations]] |
| - | | + | * [[Team:Tuebingen/NotebookReports|Reports]] |
| - | ''These page holds a detailed and technical account of the developement of our research project.''
| + | * [[Team:Tuebingen/NotebookProtocols|Protocols]] |
| - | | + | * [[Team:Tuebingen/NotebookAppendix|Appendix]] |
| - | === Preparations ===
| + | * [[Team:Tuebingen/NotebookCalendar|Calendar]] |
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| - | After determining our principal project idea we had to design our system prior to any work in the wet lab. Several steps were involved:
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| - | ==== Identification of plasmids ====
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| - | 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:
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| - | * Multiple cloning site with XbaI and SpeI restriction sites
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| - | * Multiple cloning site with beta-galactosidase, so blue-white screening is possible
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| - | * Ampicillin resistance
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| - | * amino-acid genes for selection
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| - | * Integration site for integration in the yeast genome
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| - | After consultation with Prof. Jansen, our yeast expert, we decided to use the shuttle vectors pRS313, pRS315 and pRS316.
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| - | 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.
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| - | ==== Identification of genes ====
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| - | '''Receptors:''' Aiming at aquatic environment we chose to use the membrane progesterone receptor (mPR) from the model organism ''Danio rerio'' (zebra fish). The second organism was ''Salmo salar'' (salmon) but we could not locate a homologous gene due to missing full genome sequence data. 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.
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| - | '''Signalling:''' The fet3 promoter targeted by the membrane receptors was already determined and proven working by J Smith et al. (2008). We could not obtain the sequence used by Smith and decided to take the upstream sequence (approx. 600bp) of the fet3 gene.
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| - | Since Pfet3 is regulated negatively by the mPR we decided on inverting our signal with an additional signaling step to have more sensitive measurement results. So the second part of the signalling system needs a repressor its target. Additionally, knock-out strains not containing a working repressor have to be viable. We chose the mig1 (repressor) / Psuc2 (repressor target) pair and the rox1 (repressor) / Panb1 (repressor target) pair.
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| - | '''Reporter:''' The targets of our signalling system regulate our reporter. We have access to the plasmid storage of our lab. Common reporters used in this yeast-based environment are firefly luciferase and beta-galactosidase. Both are available to us and have no legal issues concerning the publishing in the PartsRegistry.
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| - | ==== Sequence analysis and primer design ====
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| - | Next was the check of all sequences for unwanted restriction sites.
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| - | As noted above, due to several unwanted restriction sites (EcoRI and PstI) in the pRS vectors, we can only use XbaI and SpeI for assembly.
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| - | The firefly luciferase has an unwanted XbaI site so we had to use NheI/SpeI restriction instead. The XbaI site could be removed by gene synthesis.
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| - | === Weekly Reports ===
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| - | === Appendix ===
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| - | ''To complete this report we list all products and software used over the course of our project.''
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| - | ==== Chemicals ====
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| - | We needed the following chemicals:
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| - | * Ampicillin <br /> beta-lactam antibiotic
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| - | * Agarose <br /> Polysaccharide, major component of Agar
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| - | * Dimethylsulfoxid (DMSO) <br /> organic solvent
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| - | * Acetic Acid
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| - | * Ethylenediaminetetraacetic acid (EDTA)
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| - | * Ethanol
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| - | * TRIS <br /> buffer solution for enzymes
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| - | * Nucleoside triphosphate
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| - | * Trypton
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| - | * Isopropanol
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| - | * Isopropyl-β-D-thiogalactopyranosid (IPTG)
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| - | * LB-medium <br /> used for growth of E.coli
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| - | * Agar-Agar
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| - | ==== Software ====
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| - | The following list of software was used in the team:
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| - | * [http://ugene.unipro.ru/ Unipro UGENE] <br/> UGENE is a free open-source cross-platform bioinformatics software. We used it to construct and annotate all needed sequences, search for restriction sites and visualization. | + | |
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| - | * [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/vector-nti-software.html Vector NTI] (commercial) <br/> All our primers were designed using Vector NTI which is also used by our advisors. | + | |
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| - | * [http://blast.ncbi.nlm.nih.gov/Blast.cgi BLAST] <br/> BLAST was our main sequence search tool and was used for controlling of sequencing results | + | |
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| - | * [http://biologylabs.utah.edu/jorgensen/wayned/ape/ ApE] | + | |
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| - | * [http://drive.google.com Google Drive] <br/> Google Drive was used for our documentation and all of our collaborative work (papers, poster, images). | + | |
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| - | ==== Google Calendar ====
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| - | <html>
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| - | <iframe src="https://www.google.com/calendar/embed?src=4u69lnefcmvvjjvoui8fthg570%40group.calendar.google.com&ctz=Europe/Berlin" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe>
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| - | </html>
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