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From 2012.igem.org

Lab Notebook

Contents 1 Lab Notebook 1.1 Preparations 1.1.1 Identification of plasmids 1.1.2 Identification of genes 1.1.3 Sequence analysis and primer design 1.2 Weekly Reports 1.3 Software 1.4 Google Calendar

These page holds a detailed and technical account of the developement 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. We chose the mig1 (repressor) / Psuc2 (repressor target) pair and the rox1 (repressor) / Panb1 (repressor target) pair.

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.

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

Software

The following list of software was used in the team:

• [http://ugene.unipro.ru/ Unipro UGENE]
  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)
  All our primers were designed using Vector NTI which is also used by our advisors.

• [http://blast.ncbi.nlm.nih.gov/Blast.cgi BLAST]
  BLAST was our main sequence search tool and was used for controlling of sequencing results

• [http://biologylabs.utah.edu/jorgensen/wayned/ape/ ApE]

Google Calendar