Proof of concept

A number of different plasmids were assembled by the Plug ‘n’ Play system in order to verify that the system functions in mammalian cells. To demonstrate how fast any vector of choice for the expression in mammalian cells can be assembled, we designed a reporter system as proof of concept.
All the mammalian plasmids for proof of concept were constructed with the same strong constitutive cytomegalovirus (CMV) promotor, the BGH terminator, the marker cassette of Hygromycin, and the mammalian backbone, BBa_K678023. Different fluorescence reporter genes were used as the gene of interest. Furthermore, GFP was targeted to the peroxisomes to demonstrate that this reporter system is easily modified and can be suited for studies in gene expression. All transient transfections were performed in the human derived cell line U-2 OS, kindly given to us by the Danish Cancer Society.
We succeeded in proving that the Plug ‘n’ Play assembly system is easily applied for costruction of mammalian expression vectors. Transfection and expression of the fluorescence proteins GFP, mCherry, YFP, and CFP were successfully conducted in U-2 OS. Furthermore, expression of GFP targeted to the peroxisomes of U-2 OS cells succeeded. It was also shown that mammalian cells can be transfected with different fluorescence proteins at the same time. The transfected U-2 OS were fixed using 4% formaldehyde, and VECTASHIELD was added to prevent rapid loss of fluorescence while examining the cells with a confocal microscope.
This reporter system could be used for localization of proteins to specific organelles, which would allow study of the organelles in real time. Further development of this system could lead to targeting of different compartments with different fluorescence proteins at the same time.

pJEJAM1 BBa_K678049

BBa_K678049 is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescence protein is under the control of the strong constitutive CMV promoter, which can be seen in the figure below.

U-2 OS cells transiently transfected with plasmid BBa_K678049 expressing GFP. This vector provides a good expression and homogenous distribution of GFP. The white bar has a length of 20μm.	U-2 OS cells transiently transfected with pJEJAM1 expressing GFP. This vector provides a good expression and homogenous distribution of GFP. The white bar has a length of 20μm.

pJEJAM2 BBa_K678050

BBa_K678050 is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescence protein (GFP) is under the control of the strong constitutive CMV promoter (see the figure below. The peroxisomal targeting signal PTS1 is directly fused to the C-terminal of GFP, this sequence ensures the localization of GFP to the peroxisomes of the cell.

U-2 OS cells transiently transfected with plasmid BBa_K678050 expressing GFP localized to the peroxisomes. As can be seen on the picture this vector as intended localizes GFP to the peroxisomes of the cells. The white bar has a length of 30μm.

pJEJAM3 BBa_K678051

BBa_K678051 is a plasmid intended for transient transfection of mammalian cells. The expression of the yellow fluorescence protein (YFP), is under the control of the strong constitutive CMV promoter (see the figure below).

U-2 OS cells transiently transfected with plasmid BBa_K678051 expressing YFP. This vector provides a good expression and homogenous distribution of YFP. The white bar has a length of 40μm.	U-2 OS cells transiently transfected with plasmid BBa_K678051 expressing YFP. This vector provides a good expression and homogenous distribution of YFP. The white bar has a length of 30μm.

pJEJAM4 BBa_K678052

BBa_K678052 is a plasmid intended for transient transfection of mammalian cells. The expression of mCherry, a red fluorescence protein, is under the control of the strong constitutive CMV promoter (see the figure below).

U-2 OS cells transiently transfected with plasmid BBa_K678052 expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. The white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmid BBa_K678052 expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. The white bar has a length of 50μm.

pJEJAM5 BBa_K678053

BBa_K678053 is a plasmid intended for transient transfection of mammalian cells. The expression of cyan fluorescence protein (CFP), is under the control of the strong constitutive CMV promoter (see the figure below).

U-2 OS cells transiently transfected with plasmid BBa_K678053 expressing CFP. This vector provides a good expression and homogenous distribution of CFP. The white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmid pJEJAM5 expressing CFP. This vector provides a good expression and homogenous distribution of CFP. The white bar has a length of 50μm.

Mix'n'Play

Mammalian cells can be transiently transfected with several plasmids at once, allowing the simultaneous expression of different fluorescence proteins. The pictures below demonstrate different combinations of fluorescent proteins.

U-2 OS cells transiently transfected with plasmids BBa_K678050 and BBa_K678053 expressing GFP localized to the peroxisomes and CFP. The white bar has a length of 30μm.	U-2 OS cells transiently transfected with plasmids BBa_K678049 and BBa_K678053 expressing GFP and CFP. The white bar has a length of 30μm.
U-2 OS cells transiently transfected with plasmids BBa_K678049 and BBa_K678052 expressing GFP and mCherry. The white bar has a length of 70μm.	U-2 OS cells transiently transfected with plasmids BBa_K678052 and BBa_K678053 expressing YFP and CFP. The white bar has a length of 40μm.