Part:BBa_K3755007

GCaMP6m

A genetically encoded calcium indicator

Usage and Biology

GCaMP6m is a genetically encoded calcium indicator. It consists of a circularly permuted GFP (cpGFP), a calmodulin (CaM), and a calmodulin-binding peptide(CBP). Without calcium, GCaMP6m only has very low emitted light. When intracellular calcium concentration increases, emitted light at about 510nm will increase obviously. GCaMP6m is widely used to image neural activity. Our project includes a mechanosensitive cation channel, Piezo1.1 (BBa_K3755006) as the mechanosensing element. We used GCaMP6m to test whether calciums flow in cells after mechanical stimulation as we expected.

Mechanism

GCaMP6m is consists of a circularly permuted GFP (cpGFP), a calmodulin (CaM), and a calmodulin-binding peptide(CBP). Calmodulin is the only part that can respond to calcium. It has two EF-hands motifs, each EF-hands can catch two calciums. When CaM combines with four calciums, CaM will change into the activated state. Then, activated CaM will be able to attach to calmodulin-binding peptide, M13. This process will make the conformation of cpGFP tighter, which lead to an obvious increase of emitted light at about 510nm. So cpGFP plays the role of reporter. As intracellular calcium concentration increases, more GCaMP6ms can combine with calciums. So the cell which has a higher calcium concentration in its cytoplasm will be brighter under a fluorescence microscope.

Parameters

GCaMP6m has a sensitive and fast response to calcium signals.

Figure 1

Molecular cloning

We got [http://www.addgene.org/100838/ pAAV.Syn.Flex.GCaMP6m.WPRE.SV40](Part:BBa_K3755032）which contains GCaMP6m from Wei Shen'lab, Shanghaitech university. We used PCR to obtain DNA fragments of GCaMP6m. Then we inserted it behind CMV promoter using pEGFP as the backbone. Gibson assembly was used during the construction of this plasmid. The map of this plasmid is shown below.

Figure 2 Linearized pEGFP and GCaMP6m                                Map of pEGFP-GCaMP6m

Cotransfection and Stimulation

We cotransfected Piezo1.1(Part:BBa_K3755006) and GCaMP6m into HEK293 cells. GCaMP6m is a calcium indicator. If calciums flow into the cotransfected cells through Piezo1.1, a raise of green fluorescence will be observed. The following figures show that we cotransfected these two plasmids successfully.
Protocol of cotransfection
1.Dilute 12.5 μl Lipofectamine 2000 Reagent in 125 μl Opti-MEM Medium and mix thoroughly.
2.Dilute 1.25 μg plasmid1 and 1.25 μg plasmid2 in 125 μl Opti-MEM Medium and mix thoroughly.
3.Add diluted plasmid to diluted Lipofectamine 2000 Reagent (1:1 ratio).
4.Incubate for at least 5 minutes at room temperature.
5.Digest all the 293 cells which are 90% confluent in a 10 cm (diameter) cell culture dish and suspend cells with 1 ml DMEM (10%FBS).
6.Take out 200 μl cell suspension and dilute it into 5 ml with DMEM (10%FBS).
7.Slowly drop the plasmid-lipid complex into the 5 ml cell suspension and softly mix thoroughly.
8.Plate the cell suspension into 24 well cell culture dish--1 ml each well.
9.Incubate cells for 48h at 37℃.
10.Analyze transfection efficiency in use of fluorescence microscope.

Figure2:a.Red fluorescence of Piezo1.1-mRuby2, indicating the transfection of Piezo1.1    b.Green fluorescence of GCaMP after the activation of Piezo1.1 by force, 20X objective, indicating the transfection of pEGFP-GCaMP    c.Merge of the green channel and red channel, indicating the cotransfection of GCaMP and Piezo1.1

We used two different ways to stimulate Piezo1.1, and a control group is also built. The related results are shown below.
1.Yoda1 1ml 100uM Yoda1 was added to 1ml culture medium, so the final consentration of Yoda1 is 50uM. This proccess was finished under the inverted fluorescence microscope. A significant fluorescence change was observed in seconds.
Video1:Fluorescence change of cells under the stimulation of 50uM Yoda1, exposure time: 90ms, 20X objective
File:Yoda 8times.mp4
2.Shear stress We gave the HEK293 cells the shear stress by sucking out their culture medium with a pipette. The change of fluorescence is faster and more obvious than that caused by Yoda1.
Video2:Faster fluorescence change of cells under the stimulation of shear stress, exposure time: 90ms, 20X objective
File:Force 4times.mp4
3.Control In the control group, only GCaMP6m is transfected into the HEK293 cells. We also used 50uM of Yoda and shear stress to stimulate the cells in the control group. But no obvious change of fluorescence is observed in the control group under the condition of Yoda1 or shear stress.
Video3:Fluorescence change of cells in control group, exposure time: 90ms, 20X objective
File:Control 1.mp4

We also did a quantitative analysis of fluorescence changes. A program based on MATLAB was used to finish this quantitative analysis. The curve showed the characteristics of rising first and then falling under the condition of Yoda1.(Figure3a) This is important to show a decreased fluorescence intensity, which is consistent with the features of Piezo1.1. The control group showed no obvious fluorescence change.(Figure3b)

Figure3:a.Relative fluorescence of cells under the stimulation of Yoda1    b.Control group with no obvious fluorescence change

These results show that GCaMP6m indicates the changes of intracellular calcium concentration successfully.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 34
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 175
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1016
  Illegal SapI.rc site found at 922