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	== Lab Notebook ==		== Lab Notebook ==
		+	''The following pages hold a detailed and technical account of the developement of our research project.''
-	__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/NotebookAppendix|Appendix]]
-		+	* [[Team:Tuebingen/NotebookCalendar|Calendar]]
-	=== 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 (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.	+
-		+
-	'''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.	+
-		+
-	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.	+
-		+
-	'''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.	+
-		+
-		+
-	==== Sequence analysis and primer design ====	+
-		+
-	Next was the check of all sequences for unwanted restriction sites.	+
-		+
-	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.	+
-		+
-	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.	+
-		+
-		+
-	=== Weekly Reports ===	+
-		+
-		+
-	==== Week 1  (7/9-7/15) ====	+
-		+
-	Thursday the 12th of July was the first day in our laboratory.	+
-		+
-	1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.	+
-		+
-	2.  To determine the optimal annealing temperature, a gradient PCR for the parts 1-8 was performed.	+
-	Doing a gel-electrophoresis the PCR results were tested.	+
-		+
-	3. Preparing chemocompetent E. coli TOP 10 cells after Inoue protocol.	+
-		+
-		+
-	==== Week 2  (7/16-7/22) ====	+
-		+
-	1.  A successful PCR for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a gel-electrophoresis.	+
-		+
-	The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.	+
-		+
-	2. To test the competence of the chemocompetent E. coli TOP 10 cells a transformation with pRS313 and a negative control was done. Due to the fact that the competent cells didn’t work, new chemocompetent E. coli TOP 10  cells were prepared. The Transformation of pRS313, pRS315 and pRS316 in these competent cells was successful.	+
-		+
-		+
-	==== Week 3  (7/23-7/29) ====	+
-		+
-	1. The first ligation of the parts 1-8 in pGEM with following transformation in the competent E. coli TOP10 was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C.	+
-	The restriction digest was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gelelectrophoresis showed that the ligation was not successful, because only one band of 3000bp for the pGEM vector was visible.	+
-		+
-	2. A new PCR of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product 8 was purified. The PCR-product 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.	+
-		+
-		+
-	==== Week 4  (7/30-8/05) ====	+
-		+
-	1. The shipment with the synthesized parts (mPRq of Danio rerio and MIG1) arrived.	+
-		+
-	2. A transformation of the parts mPRq and MIG1 was performed using the competent E. coli TOP10 cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.	+
-	The first attempt to isolate the plasmids was with usage of a plasmid preparation kit. But this try failed. Therefore the plasmid isolation was successfully repeated using the alkaline lysis.	+
-		+
-	3. A new PCR of the parts 1-8 with Taq/Pfu-polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.	+
-		+
-		+
-	==== Week 5  (8/06-8/12) ====	+
-		+
-	1. A small restriction digest of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the right overhangs for a ligation to take place later.	+
-	The restriction digest was executed with the parts Mig1 and mPR of Danio rerio, too.	+
-	Due to unclean plasmids and DNA (perhaps to much salts in the tubes) this step had to be repeated several times, because the restriction digest beeing incomplete.	+
-		+
-	Therefore the plasmids (pRS313, 315, 316 and the parts Mig1, mPR D.R.) were purified again with a midi DNA purification kit. Now the restriction digest was executed completely.	+
-	A mini plasmid preparation was performed afterwards to purify DNA in order to prepare the DNA for ligation.	+
-		+
-	2. A new PCR of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR-product. The polymerase Herculase was used due to its preciseness and productivity. Indeed the result of the PCR was better than with Pfu/Taq-polymerase.	+
-		+
-	A Preparative gel for PCR products 3,4,5,6,7,8 (from PCR with Herculase) delivered new template for another PCR with Taq/Pfu Polymerase.	+
-		+
-		+
-	==== Week 6  (8/13-8/19) ====	+
-		+
-	1. The first successful ligation and transformation into pGEM vector of part 4 in E. Coli TOP10 was executed. A lot of colonies grew on the agar-plate. After a restriction digest with XbaI and SpeI and the control with a gel-electrophoresis the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected DNA-sequence.	+
-	A midi-prep and restriction digest followed in order to prepare them for later ligation in pRS vectors.	+
-		+
-	2. Ligation of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C (39.2 Fahrenheit). The transformation of these parts was executed into E. coli TOP10.	+
-	After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3,6,7,8 did not work,  we had to get back to the restriction digest. Positive samples were prepared for sequencing. The Parts 3 and 8 were sequenced successfully. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as promotor for luciferase.	+
-		+
-	3. We received the synthesized receptor of Xenopus laevis. It was successfully transformed in E. coli TOP10 and purified with midi-prep.	+
-		+
-		+
-	==== Week 7  (8/20-8/26) ====	+
-		+
-	1. The PCR of the parts 1 and 2 with Herculase polymerase was executed.	+
-	The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.	+
-		+
-	2. Since we ran out of luciferase DNA, we decided to transform remaining DNA of luciferase into E.coli TOP10. The transformation was successful.	+
-		+
-	3. The receptors (mPR Danio rerio, mPR Xenopus laevis) and mig1 were initially ligated into pRS vectors and transformed into E.coli (TOP10). But there were no colonies on the agar-plate.	+
-		+
-		+
-	==== Week 8  (8/27-9/02) ====	+
-		+
-	==== Week 9 (9/03-9/09) ====	+
-		+
-	==== Week 10 (9/10-9/16) ====	+
-		+
-	==== Week 11 (9/17-9/23) ====	+
-		+
-	==== Week 11 (9/24-9/30) ====	+
-		+
-	=== Appendix ===	+
-		+
-	''To complete this report we list all products and software used over the course of our project.''	+
-		+
-	==== Chemicals ====	+
-		+
-	We needed the following chemicals:	+
-		+
-	* Ampicillin <br /> beta-lactam antibiotic	+
-		+
-	* Agarose <br /> Polysaccharide, major component of Agar	+
-		+
-	* Dimethylsulfoxid (DMSO) <br /> organic solvent	+
-		+
-	* Acetic Acid	+
-		+
-	* Ethylenediaminetetraacetic acid (EDTA)	+
-		+
-	* Ethanol	+
-		+
-	* TRIS <br /> buffer solution for enzymes	+
-		+
-	* Nucleoside triphosphate	+
-		+
-	* Trypton	+
-		+
-	* Isopropanol	+
-		+
-	* Isopropyl-β-D-thiogalactopyranosid (IPTG)	+
-		+
-	* LB-medium <br /> used for growth of E.coli	+
-		+
-	* Agar-Agar	+
-		+
-		+
-		+
-	==== Software ====	+
-		+
-	The following list of software was used in the team:	+
-	* [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.	+
-		+
-	* [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.	+
-		+
-	* [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	+
-		+
-	* [http://biologylabs.utah.edu/jorgensen/wayned/ape/ ApE]	+
-		+
-	* [http://drive.google.com Google Drive] <br/> Google Drive was used for our documentation and all of our collaborative work (papers, poster, images).	+
-		+
-		+
-	==== Google Calendar ====	+
-		+
-	<html>	+
-	<iframe src="https://www.google.com/calendar/embed?src=4u69lnefcmvvjjvoui8fthg570%40group.calendar.google.com&amp;ctz=Europe/Berlin" style="border: 0" width="800" height="600" frameborder="0" scrolling="no"></iframe>	+
-	</html>	+

---

Revision as of 09:46, 13 September 2012

Lab Notebook

The following pages hold a detailed and technical account of the developement of our research project.

• Preparations
• Reports
• Appendix
• Calendar