"

From 2012.igem.org

Preparations

Contents 1 Preparations 1.1 Identification of plasmids 1.2 Identification of genes 1.3 Sequence analysis and primer design

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.

Three weeks before the official lab time started, we designed our primers with [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/vector-nti-software.html Vector NTI]. The programm was basically used to compute good primers. We then manually optimized the primer sequences by setting G and C nucleotides at the 3'-tail so that the development of hydrogen bridge bonds is increased, which leads to stronger bindings. In addition the primer sequences were modified so that the related primer pairs have preferably the same or close annealing temperatures.

Part #	Part name	forward primer	reverse primer
1	lacZ	5’-GCTAGCATGGTGCTGCGTTGG	5’-ACTAGTTTATTTTTGACACCAG
2	luciferase	5’-GCTAGCATGGAAGACGCCAAA	5’-ACTAGTTTAAAGCTTCTTTCCGCC
3	Padh1	5’-TCTAGAAAGAAATGATGGTAAA	5’-ACTAGTAGTTGATTGTATGCTT
4	Psuc2	5’-TCTAGACATACTAAGACATTTACCG	5’-ACTAGTCATATACGTTAGTGAAAA
5	Pfet3	5’-TCTAGACATTACTGCTGTAAAAAGG	5’-GCAAAAAATTAGAACTAGACTAGT
6	Panb1	5’-TCTAGATTTTTTCCTGTGTTCACC	5’-ACTAGTGTTTTAGTGTGTGAATGA
7	Tadh1	5’-TCTAGAGCGAATTTCTTATGATTT	5’-ACTAGTAGGTGTTGTCCTCTGAGG
8	rox1	5’-TCTAGAATGAATCCTAAATCCTCTACAC	5-ACTAGTAATTGTTCTTTTGAGGCG