Part:BBa_K363006

A characterized calcium dependent response element binding site for the Crz1 activator

This part is a binding site for the Crz1 transcriptional activator. When placed upstream (~700 bp in the yeast gene FKS2/GSC2) of a eukaryotic promoter sequence, 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.

Usage and Biology

This plot shows a the increasing linear relationship between electrostimulation duration and expression of the RFP reporter, controlled by the promoter incorporating FKS2-CDRE UAS.

This part was characterized as part of a larger sequence (see design notes), 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. For full details, see the 2010 Johns Hopkins Team wiki.

FKS2 CDRE with vesicular pumps, 0 seconds shocking

FKS2 CDRE with vesicular pumps, 90 seconds shocking

FKS2 CDRE with vesicular pumps, 110 seconds shocking

In the images above we observe some constitutive expression of RFP in the FKS2 CDRE strain with the vesicular pumps. However, there is a linear increase in expression of the RFP reporter with increase in electrostimulation time up to 90 seconds.

FKS2 CDRE without vesicular pumps, 80 seconds shocking

FKS2 CDRE without vesicular pumps, 90 seconds shocking

FKS2 CDRE without vesicular pumps, 110 seconds shocking

FKS2 CDRE without vesicular pumps, 130 seconds shocking

Again, there is a linear relationship between stimulation duration and RFP reporter expression. However, in this case, the cells lacking vesicular pumps expressed less than those with vesicular pumps, and were less stable under electrostimulatory conditions.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]