Part:BBa_K387012

CRE-JeT promoter

This part is an alignment of CRE (cAMP response element) enhancer and JeT core promoter which is sensitive to calcium signals in eukaryotic cells. CRE is the binding site of CRE-BP transcription factor family and JeT promoter is a synthetic core promoter which recruits basal transcription machinery.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal suffix found in sequence at 472
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 251
1000
COMPATIBLE WITH RFC[1000]

Characterization of CRE-JeT promoter

By Han Zhu, Yueling Zhou, Yuan Liu & Chuan Shan from iGEM2010_SJTU-BioX-Shanghai

The thought and method for the test of CRE-Jet part is similar to that of MEF2-JeT. The only difference between them is that in this test the enhancer before Jet is replaced by CRE, so we would turn to results directly.

1. Work as a good promoter

Result: As we expected, expression level of luciferase in experimental group transfected with CRE-Jet-luciferase largely outgrows its counterpart with pGL3-Basic. The picture below well depicts the function of CRE-Jet as a good promoter. However, by comparing the experimental groups varying in Ionomycin concentration, we get no favorable evidence in explaining calcium ions could regulate the enhancer-promoter. In theory, after adding 5μM Ionomycin, it would have a significant intracellular increase of Ca2+, which would show a great up-regulated expression of luciferase.

In order to explain the result, we searched for more research and studies and find out that there is calcium in the cell culture (proximally 2mM) and that concentration might be enough to start transcription. Then we come up with the idea to reduce the calcium concentration using EGTA (ethylene glycol tetraacetic acid) a polyamino carboxylic acid, a chelating agent that is related to the better known EDTA, but with a much higher affinity for calcium. The result will be shown below.

2. Promoter functions without Ca2+ from medium

Result: The figure below strongly demonstrated that CRE-JeT promoter has dramatically promotes the level of expression with the stimuli of calcium/Ionomycin, compared with the control group (whose Ca2+ concentration is reduced to almost zero with the help of EGTA).

While there is still the problem that the expression level of luciferase when there is no calcium is relatively high. Similarly, we tried to figure out the reason for this phenomenon by referring to the mechanism of the CRE enhancer. As is shown in the project page, there is no research showing that the CRE enhancer has the function of blocking transcription. And the activation function is based on epigenetical mechanism as well. In that case, the unexpected transcription initiation is the result of JeT core promoter.