Part:BBa_K387011

MEF2-JeT promoter

This part is an alignment of MEF2 enhancer and JeT core promoter, and is used to sense calcium signals. MEF2 enhancer is the binding site of transcription factor MEF2c, which can recruit other factors such as HDAC and Cabin1 and block transcription. The promoter is activated when calcium influx increases because Ca2+ could release Cabin1 and HDAC. 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 MEF2-JeT promoter

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

1. Work as a good promoter

Idea: In order to test whether MEF2-Jet could function as a good promoter, we set two groups, experimental and control, to verify it. We reconstructed pcDNA3.1 by connecting MEF2-Jet with the gene encoding luciferase. So, if MEF2-Jet could work normally, the expression of luciferase will gain a huge increase than that with no promoter in its upstream.

Method: Cultured ECHO and C3H10 cells in 24-well plate for 24h (medium for C3H10: DMEM, 10% FBS and 1% Penicillin/Streptomycin). Experimental groups were transfected with pcDNA3.1 containing MEF2-Jet-luciferase, while control groups were transfected with pGL3-Basic which contains no promoter. After another 24h, we conducted a dual-luciferase assay to measure the expression of luciferase.

Note: We have searched related information and known the calcium concentration of the medium for C3H10 is 2mmol/L, because of the calcium ions got from DMEM and FBS.

Result: As we expected, expression level of luciferase in experimental group transfected with MEF2-Jet-luciferase largely outgrows its counterpart with pGL3-Basic. The picture below well depicts the function of MEF2-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 found 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

Idea: As the medium we use to culture C3H10 is DMEM mixed with 10% FBS, the Ca2+ concentration in this medium is about 2mM, which is not negligible in our experiment. In order to make clear whether the Ca2+ in the medium could activate the MEF-Jet circuit or not, we need to eliminate all dissolved Ca2+ cation and then measure the activity of luciferase.

Method: EGTA(ethylene glycol tetraacetic acid) has a high affinity for calcium so we use it as a kind of calcium chelator to eliminate the Ca2+ in the medium. In our experiment, EGTA was added into medium 8h after transfection with MEF-Jet. The experimental groups was added EGTA while the control ones not. Dual-Luciferase assay was conducted 24h after transfection.

Result: After treated with EGTA, the expression activity of luciferase shows slightly increase with the increase of the concentration of Ionomycin (shown in figure below column marked A, B and C). However, the increase of expression level is not as high as we have expected because column a, who is supposed to have no luciferase expression showed high level fluorescence.

To explain the result, we need to look carefully into the mechanism of MEF2 enhancer. As is explained in the project page, MEF2 enhancer who recruits series of transcription factors regulates the activity of transcription mainly by epgenetically process (MEF2 recruits HDACs to down regulate transcription and p300 HAT to up regulate it). However, in our experiments, we use pcDNA as our vector, which will not be integrated into the cell chromosome and the system it contains will not be modified by nucleosome. Therefore, the transcription activity has less relationship with the stimuli of ionomycin. Moreover, as a synthetic core promoter, JeT promoter has relatively high level of transcription activity, that result in the luciferase expression when there is no calcium (column a, with EGTA added and no ionomycin introduced).

This problem will be solved if we use viral vectors instead of pcDNA, however, viral transfection and cell culture methods takes much more time than our current method and we did not have time to finish these experiments before the deadline when wiki freezes.