Team:Johns Hopkins/Parts

From 2010.igem.org

JHU.

Contents

Parts

BBa_K363001-BBa_K363007

Calcium dependent response elements (binding sites) for the Crz1 activator.

This part is a binding site for the Crz1 transcriptional activator. When placed upstream of a eukaryotic promoter sequence (~700bp in the case of FKS2), this sequence results in the calcium dependant transcription of downstream coding sequences given that Crz1 and the upstream signal transducing phosphotase calcineurin are constitutively expressed. The signal cascade occurs when calcineurin binds to cytosolic calcium, activating its phosphotase activity. The active calcineurin dephosphorylates cytoplasmic Crz1, resulting in translocation of Crz1 to the nucleus where it binds to this part and promotes the assembly of general transcription factors at the promoter sequence.

BBa_K363001

The consensus calcium dependent response element binding site for the Crz1 activator.
This part was derived from calcium dependent response element (CDRE) binding sequence of the FKS2 gene. The consensus sequence for Crz1 binding as described by Yoshimoto et al (2002) was mutated at non conserved residues to generate this part. This UAS is the consensus sequence for Crz1 binding, and is expected to have higher binding than the UAS characterized in BBa_K363006, as it is the consensus sequence and occurs with ~4 times higher frequency than the characterized sequence in the yeast genome.

BBa_K363006

A characterized calcium dependent response element binding site for the Crz1 activator.
This part was characterized as part of a larger sequence (see design notes for this part in the registry), as it occurs naturally as an activator of the yeast gene FKS2, which is codes for an enzyme involved in spore wall synthesis. We found that applying a voltage of 8V for 40-80 seconds provided maximal induction using our apparatus. This apparatus was, roughly speaking, a cylindrical coaxial electrode that was about .75cm across. We used SC media.

BBa_K363008 and BBa_K363009

The voltage-gated calcium channels of S. cerevisiae.
The channels, Mid1 and Cch1, are used implicitly throughout the project, as they are the primary mechanism of voltage sensitivity in S. cerevisiae. These channels were used by both our team and by Valencia in 2009. Valencia posted null mutants of these genes in 2009, but we thought that it would be useful to have the wild-type sequences in the registry.

BBa_K363010, BBa_K363011, and BBa_K363012

The calcium stress response system in S. cerevisiae.
These genes, Crz1, Cna1, and Cnb1, are used implicitly throughout our project, as this is the machinery that yeast use to respond to their intrinsic calcium sensitivity. Cna1 is the catalytic subunit of calcineurin, and Cnb1 is the regulatory subunit of calcineurin. This protein phosphatase acts on Crz1 in the presence of calcium. Crz1 is a widely acting transcription factor that binds to a conserved binding site in the yeast genome (see parts BBa_K363001-BBa_K363007).

Registry Parts

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