Difference between revisions of "Part:BBa K2295001"

Latest revision as of 00:57, 2 November 2017

CRE (cAMP response element)

Being one of the main downstream signaling pathways of Gs coupled G protein-coupled receptors (GPCRs), this BioBrick supports the combination with a wide range of other parts. The iGEM BioBrick library already contains several Gs coupled GPCRs; depending on the GPCR, different inputs can be used for ligand dependent gene expression.

G protein coupled receptors in general

Figure 1:

TDAG8 signaling pathway

GPCRs, also known as heptahelical receptors, are a large family of integral membrane proteins that respond to many different extracellular stimuli. The two principal signal transduction pathways involving GPCRs are the cAMP and the phosphatidylinositol signaling pathways.

Mechanism

Due to the fact that this BioBrick is mainly used for cAMP dependent transcription, only the cAMP cascade will be described, which is characteristical for Gs coupled GPCRs. Being activated by low extracellular pH, a conformational change takes place in the receptor. This change is transmitted to an attached intracellular heterotrimeric G protein complex (Figure 1: Gs). Exchanging GDP for GTP due to the stimulation, the Gs alpha subunit is released from the complex (not depicted in Figure 1). Binding to adenylyl cyclase (AC), Gs alpha subunit activates AC. This results in the catalyzation of the conversion of ATP into cyclic adenosine monophosphate (cAMP). Although the increase of intracellular concentration of this secondary messenger has numerous effectors, the main pathway of BBa_K2295001 continues with the activation of the cAMP dependent enzyme protein kinase A (PKA). PKA phosphorylates a number of other proteins. It also translocates into the nucleus where it activates cAMP responsive binding elements (CREB). Being now phosphorylated, CREBs can bind to cAMP responsive element (CRE) (BBa_K2295001), activating downstream transcription (Fig. 1).

Freiburg 2017's Promoter characterization

In order to characterize the CRE, stably transduced HEK293T and Jurkat lines were created expressing eCFP under a minimal promoter with multiple CREs. Induction was performed with pH adjusted media. Constitutively expressed mCherry was used as transduction marker. For analysis in HEK293T a PEI transfection of TDAG8, which is not expressed in these cells, was performed (Ausländer et al., 2014). To generate a high expression by activating the signaling cascade downstream of the receptor, the stable cell lines were induced with forskolin and IBMX. Forskolin activates the cAMP-producing enzyme adenylyl cyclase and IBMX inhibits cAMP-hydrolyzing phosphodiesterases (Bittinger et al., 2004). Fluorescence was measured by flow cytometry after 24 h of incubation (Fig. 2).

Figure 2: Flow cytometry of hypoxia response element promoter analysis. a) Jurkat cells stably transduced with 4xCRE-pTal:eCFP were incubated 24 h in pH adjusted RPMI 1640. b) The experiment was repeated inducing with Forskolin (10 µM) and IBMX (10 µM). c) and d) HEK293T cells stably transduced with 4xCRE-pTal:eCFP with and without transient TDAG8 were induced similarly to a) and b). Results show the represent the amount of eCFP positive cells c) and the d) mean fluorescence intensity. All data points are mean values of triplicates, error bars represent standard deviation. Significant differences in a) and b) were determined using ANOVA, for c) and d) significant differences were determined using one-tailed student’s t-test (Excel 2017); * p < 0.05, ** p < 0.01, *** p < 0.001, non-significant differences are not marked.

Sequencing Results Freiburg 2017

Figure 3: Sequencing Results

Sanger sequencing was done at GATC with BBa_G00101. Geneious was used to map result to the expceted sequence.

Sequence and Features

References

Ausländer, D. et al.. A synthetic multifunctional mammalian pH sensor and CO2 transgene-control device. Mol. Cell 55, 397–408 (2014).

Bittinger, M. A. et al Activation of cAMP Response Element-Mediated Gene Expression by Regulated Nuclear Transport of TORC Proteins. Current Biology, Vol. 14, 2156–2161 (2004).