Latest revision as of 00:24, 22 September 2011

Proof of concept in mammalian cells

Proof of concept

Five different plasmids were assembled with the Plug ‘n’ Play standard 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.

The plasmids for proof of concept in mammalian cells were constructed with the strong constitutive cytomegalovirus (CMV) promotor, the BGH terminator, the hygromycin marker cassette, and the plasmid backbone pMam. Different genes encoding fluorescent proteins were used as the gene of interest, and the reporter system can easily be modified to be used for gene expression analysis. All transient transfections were performed in the human derived cell line U-2 OS, kindly provided by the Danish Cancer Society. For microscopy the transfected U-2 OS cells were fixed using 4% formaldehyde, and VECTASHIELD was added to prevent rapid loss of fluorescence while examining the cells with the confocal microscope.

We have proved that the Plug ‘n’ Play assembly standard can easily be applied for construction of mammalian expression vectors. Transfections and expression of the fluorescent proteins GFP, mCherry, YFP, and CFP were successfully conducted in U-2 OS cells. Furthermore, expression of GFP targeted to the peroxisomes of U-2 OS cells succeeded. Lastly it was shown that mammalian cells can be transfected with different fluorescent proteins simultaneously.

This reporter system could be further developed enabling the localization of proteins to specific organelles. Such a system would allow the study of organelles and other compartments in real time.

pJEJAM1 BBa_K678049

pJEJAM1 is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescent protein (GFP) 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 pJEJAM1 expressing GFP. This plasmid 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 plasmid provides a good expression and homogenous distribution of GFP. The white bar has a length of 20μm.

pJEJAM2 BBa_K678050

pJEJAM2 is a plasmid intended for transient transfection of mammalian cells. The expression of 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 should ensure the localization of GFP to the peroxisomes of the cell. Although the microscopy shows GFP distributed in points, further analysis are necessary to confirm that GFP is in fact is targeted to the peroxisomes.

U-2 OS cells transiently transfected with pJEJAM2 expressing GFP localized to the peroxisomes. As can be seen on the picture this vector most likely localizes GFP to the peroxisomes of the cells. The white bar has a length of 30μm.

pJEJAM3 BBa_K678051

pJEJAM3 is a plasmid intended for transient transfection of mammalian cells. The expression of the yellow fluorescent protein (YFP), is under the control of the strong constitutive CMV promoter (see the figure below).


U-2 OS cells transiently transfected with pJEJAM4 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 pJEJAM4 expressing YFP. This vector provides a good expression and homogenous distribution of YFP. The white bar has a length of 30μm.

pJEJAM4 BBa_K678052

pJEJAM4 is a plasmid intended for transient transfection of mammalian cells. The expression of mCherry, a red fluorescent protein, is under the control of the strong constitutive CMV promoter (see the figure below).


U-2 OS cells transiently transfected with pJEJAM4 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 pJEJAM4 expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. The white bar has a length of 50μm.

pJEJAM5 BBa_K678053

pJEJAM5 is a plasmid intended for transient transfection of mammalian cells. The expression of cyan fluorescent protein (CFP), is under the control of the strong constitutive CMV promoter (see the figure below).


U-2 OS cells transiently transfected with pJEJAM5 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 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 fluorescent proteins. The pictures below demonstrate different combinations of fluorescent proteins.


U-2 OS cells transiently transfected with pJEJAM2 and pJEJAM5 expressing GFP localized to the peroxisomes and CFP, respectively. The white bar has a length of 30μm.	U-2 OS cells transiently transfected with pJEJAM1 and pJEJAM5 expressing GFP and CFP, respectively. The white bar has a length of 30μm.

U-2 OS cells transiently transfected with pJEJAM1 and pJEJAM4 expressing GFP and mCherry, respectively. The white bar has a length of 70μm.	U-2 OS cells transiently transfected with pJEJAM3 and pJEJAM5 expressing YFP and CFP, respectively. The white bar has a length of 40μm.

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-<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Mammalian cells" class="h1"> Mammalian cells</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Proof of concept" class="h1"> Proof of concept</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Device BBa_K678049" class="h2"> pJEJAM1 <br>
+BBa_K678049</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Device BBa_K678050" class="h2"> pJEJAM2 <br>
+BBa_K678050</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Device BBa_K678051" class="h2"> pJEJAM3 <br>
+BBa_K678051</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Device BBa_K678052" class="h2"> pJEJAM4 <br>
+BBa_K678052</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Device BBa_K678053" class="h2"> pJEJAM5 <br>
+BBa_K678053</a><br><br>
+<a href="https://2011.igem.org/Team:DTU-Denmark-2/results/Proofofconcept/mammalian#Play'n'Mix" class="h2"> Mix'n'Play</a><br><br>
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-<a name="Mammalian cells"></a><h1><b>Mammalian cells</b></h1>
+<a name="Proof of concept"></a><h1><b>Proof of concept</b></h1>
 <p align="justify">
-Why Mammalian cells? - Mammalian cells as cell factories<br>
+Five different plasmids were assembled with the Plug ‘n’ Play standard 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. <br><br>
-The potential in biopharmaceuticals produced by mammalian cells is huge (1). Last year the global biopharmaceutical market in 2015 was expected to increase to US$ 167 billion (2). The biopharmaceuticals target diseases such as psoriasis and cancer and are more convenient, safe and effective than the other treatment solutions on the market (3). Compared to microbes mammalian cells have lower yields, are more difficult to handle in the laboratory, are costly and have a slow doubling time. So why use mammalian cells, when microbes such as yeast and E. coli are already used as fast, efficient, and cheap cell factories? <br>
+The plasmids for proof of concept in mammalian cells were constructed with the strong constitutive <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_J52034">cytomegalovirus (CMV) promotor</a>, the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678019">BGH terminator</a>, the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678021">hygromycin marker cassette</a>, and the plasmid backbone <a href="http://partsregistry.org/Part:BBa_K678023">pMam</a>.
-The main and most important answer to this is post-translational modifications (PTMs) (4). The reasons for the importance of the PTMs are the protein stability, ligand binding, and potential result in increased immunogenicity when used for humans (4). Most of the therapeutic proteins approved and currently in development are post-translationally modified, which is not surprising since approximately 50% of human proteins are glycosylated (5,5). <br><br>
+Different genes encoding fluorescent proteins were used as the gene of interest, and the reporter system can easily be modified to be used for gene expression analysis. All transient transfections were performed in the human derived cell line U-2 OS, kindly provided by the Danish Cancer Society. For microscopy the transfected U-2 OS cells were fixed using 4% formaldehyde, and VECTASHIELD was added to prevent rapid loss of fluorescence while examining the cells with the confocal microscope.<br><br>
+We have proved that the Plug ‘n’ Play assembly standard can easily be applied for construction of mammalian expression vectors. Transfections and expression of the fluorescent proteins GFP, mCherry, YFP, and CFP were successfully conducted in U-2 OS cells. Furthermore, expression of GFP targeted to the peroxisomes of U-2 OS cells succeeded. Lastly it was shown that mammalian cells can be transfected with different fluorescent proteins simultaneously. <br><br>
-With this kept in mind there are some key issues in manufacturing where speed in development plays a prominent role. Cell line creation has to be complete as rapidly as possible and to reduce the timelines for cell line creation the Plug’n’Play with DNA assembly standard could be a valuable tool.<br><br>
+This reporter system could be further developed enabling the localization of proteins to specific organelles. Such a system would allow the study of organelles and other compartments in real time.
-It is not only in the industry a more efficient high throughput cloning system could be valuable, also when it comes to constructing DNA libraries from extremely complex DNA populations such as total human genomic DNA it would be useful.<br><br>
-To demonstrate how fast you can assemble any vector of choice for the expression in mammalian cells we chose to design a reporter system as proof of concept. This system comprises a backbone plasmid that allows for the amplification in and selection of <i>E. coli</i>, the strong constitutive promoter CMV, a gene module consisting of a gene encoding a fluorescent protein or a gene encoding a fluorescent protein and a localization signal, the BGH terminator and the hygromycin marker cassette. On this page we present the results of the reporter system.
-In total we spent blabla days on constructing, blabla days on transfecting and went from design to result in blabla days
 </p>
-<br>
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-<a name="pJEJAM1"></a><h2>pJEJAM1</h2>
+<a name="Device BBa_K678049"></a><h2>pJEJAM1 BBa_K678049</h2>
-The results shows fluorescence spread evenly in the hyphae which correlate with that in device BBa_K678060, the gene of interest, only consists of a green fluorescence protein GFP with no specific target.
+<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678049">pJEJAM1</a> is a plasmid intended for transient transfection of mammalian cells. The expression of the green fluorescent protein (GFP) is under the control of the strong constitutive CMV promoter, which can be seen in the figure below.
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-<td><img src="https://static.igem.org/mediawiki/2011/1/1b/IGEM_001.tif" height="250px" align="center"> </img></td>
+<td><img src="https://static.igem.org/mediawiki/2011/5/53/IGEM_001.png" height="250px" align="center"> </img></td>
-<td><img src="https://static.igem.org/mediawiki/2011/f/f6/IGEM3.tif" height="250px" align="center"> </img></td>
+<td><img src="https://static.igem.org/mediawiki/2011/4/4a/IGEM3.png" height="250px" align="center"> </img></td>
 </tr>
 <tr>
-<td>U-2 OS cells expressing plasmid pJEJAM1 </td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678049">pJEJAM1</a> expressing GFP. This plasmid provides a good expression and homogenous distribution of GFP. The white bar has a length of 20μm. </td>
-<td>U-2 OS cells expressing plasmid pJEJAM1</td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678049">pJEJAM1</a> expressing GFP. This plasmid provides a good expression and homogenous distribution of GFP. The white bar has a length of 20μm. </td>
 </tr>
 </table>
+<br><br>
+<a name="Device BBa_K678050"></a><h2>pJEJAM2 BBa_K678050</h2>
+<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678050">pJEJAM2</a> is a plasmid intended for transient transfection of mammalian cells. The expression of 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 should ensure the localization of GFP to the peroxisomes of the cell. Although the microscopy shows GFP distributed in points, further analysis are necessary to confirm that GFP is in fact is targeted to the peroxisomes.
+<br>
+<br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2011/3/3c/Devices_12.1.png" height="110px" align="center"> </img>
+<br>
 <table cellpadding="10px" align="center">
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-<td><img src="https://static.igem.org/mediawiki/2011/1/12/22-7_back.jpeg" height="250px" align="center"> </img></td>
+<td><center><img src="https://static.igem.org/mediawiki/2011/b/b1/12-1%2B16_034.png" height="250px" align="center"> </img></center></td>
-<td><img src="https://static.igem.org/mediawiki/2011/8/8e/22-7_back_rettet.jpg" height="250px" align="center"> </img></td>
 </tr>
 <tr>
-<td><i>Aspergillus nidulans </i> with device BBa_K678060 - Front</td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678050">pJEJAM2</a> expressing GFP localized to the peroxisomes. As can be seen on the picture this vector most likely localizes GFP to the peroxisomes of the cells. The white bar has a length of 30μm. </td>
-<td><i>Aspergillus nidulans </i> with device BBa_K678060 - Back </td>
 </tr>
 </table>
-</p>
+<br><br>
+<a name="Device BBa_K678051"></a><h2>pJEJAM3 BBa_K678051</h2>
+<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678051">pJEJAM3</a> is a plasmid intended for transient transfection of mammalian cells. The expression of the yellow fluorescent protein (YFP), is under the control of the strong constitutive CMV promoter (see the figure below).
 <br>
 <br>
-<br>
+<p align="center">
-<br>
+<img src="https://static.igem.org/mediawiki/2011/f/f6/Device14.png" height="110px" align="center"> </img>
-<img src="https://static.igem.org/mediawiki/2011/3/3c/Devices_12.1.png" height="125px" align="center"> </img>
-<br>
-<br>
-<br>
 <br>
-<br>
+<table cellpadding="10px" align="center">
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-<br>
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-<img src="https://static.igem.org/mediawiki/2011/f/f6/Device14.png" height="125px" align="center"> </img>
+<tr>
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+<td><img src="https://static.igem.org/mediawiki/2011/d/d3/14%2B16_gul.png" height="250px" align="center"> </img></td>
-<br>
+<td><img src="https://static.igem.org/mediawiki/2011/e/e3/1_14_gul.png" height="250px" align="center"> </img></td>
-<br>
+</tr>
-<br>
+<tr>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678051">pJEJAM4</a> expressing YFP. This vector provides a good expression and homogenous distribution of YFP. The white bar has a length of 40μm. </td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678051">pJEJAM4</a> expressing YFP. This vector provides a good expression and homogenous distribution of YFP. The white bar has a length of 30μm. </td>
+</tr>
+</table>
+<br><br>
+<a name="Device BBa_K678052"></a><h2>pJEJAM4 BBa_K678052</h2>
+<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678052">pJEJAM4</a> is a plasmid intended for transient transfection of mammalian cells. The expression of mCherry, a red fluorescent protein, is under the control of the strong constitutive CMV promoter (see the figure below).
 <br>
 <br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2011/f/f6/Device_15.png" height="110px" align="center"> </img>
-<img src="https://static.igem.org/mediawiki/2011/f/f6/Device_15.png" height="125px" align="center"> </img>
-<br>
-<br>
-<br>
-<br>
 <br>
 <br>
-<img src="https://static.igem.org/mediawiki/2011/4/4c/Device_16.png" height="125px" align="center"> </img>
+<table cellpadding="10px" align="center">
+<tr>
+<td><img src="https://static.igem.org/mediawiki/2011/c/cf/5_rød-2.png" height="250px" align="center"> </img></td>
+<td><img src="https://static.igem.org/mediawiki/2011/1/16/26.08.png" height="250px" align="center"> </img></td>
+</tr>
+<tr>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678052">pJEJAM4</a> expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. The white bar has a length of 70μm. </td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678052">pJEJAM4</a> expressing mCherry. This vector provides a good expression and homogenous distribution of mCherry. The white bar has a length of 50μm. </td>
+</tr>
+</table>
+<br><br>
+<a name="Device BBa_K678053"></a><h2>pJEJAM5 BBa_K678053</h2>
+<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678053">pJEJAM5</a> is a plasmid intended for transient transfection of mammalian cells. The expression of cyan fluorescent protein (CFP), is under the control of the strong constitutive CMV promoter (see the figure below).
 <br>
 <br>
+<p align="center">
+<img src="https://static.igem.org/mediawiki/2011/4/4c/Device_16.png" height="110px" align="center"> </img>
-<br>
 <br>
-References<br><br>
+<table cellpadding="10px" align="center">
-(1)	Hesse, F., Wagner, R. (2000). Developments and improvements in the manufacturing of human therapeutics with mammalian cell cultures. TIBTECH, 18, 173-180.<br><br>
-(2)	Global Biopharmaceutical Market Report (2010-2015). (2010). The International Market Analysis Research and Consulting Group.<br><br>
+<tr>
+<td><img src="https://static.igem.org/mediawiki/2011/0/0d/16_033.png" height="250px" align="center"> </img></td>
+<td><img src="https://static.igem.org/mediawiki/2011/9/9e/16_blå.png" height="250px" align="center"> </img></td>
+</tr>
+<tr>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678053">pJEJAM5</a> expressing CFP. This vector provides a good expression and homogenous distribution of CFP. The white bar has a length of 70μm. </td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678053">pJEJAM5</a> expressing CFP. This vector provides a good expression and homogenous distribution of CFP. The white bar has a length of 50μm.  </td>
+</tr>
+</table>
-(3)	Xie, L., Zhou, W., Robinson, D. (2003). Protein production by large-scale mammalian cell culture. S.C. Makrides (Ed.) Gene Transfer and Expression in Mammalian Cells. Elsevier Science B.V.<br><br>
+<br><br>
+<a name="Play'n'Mix"></a><h2>Mix'n'Play </h2>
+Mammalian cells can be transiently transfected with several plasmids at once, allowing the simultaneous expression of different fluorescent proteins. The pictures below demonstrate different combinations of fluorescent proteins.
+<br>
+<br>
-(4)	Browne, SM., Al-Rubeai, M. (2007). Selection methods for high-producing mammalian cell lines. TRENDS in Biotechnology, 25, 425-432.<br><br>
+<table cellpadding="10px" align="center">
-(5)	Walsh, W., Jefferis, R. (2006). Post-translational modifications in the context of therapeutic proteins. Nature Biotechnology, 24, 1241-1252.<br><br>
+<tr>
+<td><img src="https://static.igem.org/mediawiki/2011/7/78/Grøn_%2Bblå.png" height="250px" align="center"> </img></td>
+<td><img src="https://static.igem.org/mediawiki/2011/9/99/16_blå%2Bgrøn.png" height="250px" align="center"> </img></td>
+</tr>
+<tr>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678050">pJEJAM2</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678053">pJEJAM5</a> expressing GFP localized to the peroxisomes and CFP, respectively. The white bar has a length of 30μm. </td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678049">pJEJAM1</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678053">pJEJAM5</a> expressing GFP and CFP, respectively. The white bar has a length of 30μm. </td>
+</tr>
+<tr>
+<td><img src="https://static.igem.org/mediawiki/2011/1/10/1%2B15_033.png" height="250px" align="center"> </img></td>
+<td><img src="https://static.igem.org/mediawiki/2011/6/6b/14%2B16_033.png" height="250px" align="center"> </img></td>
+</tr>
+<tr>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678049">pJEJAM1</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678052">pJEJAM4</a> expressing GFP and mCherry, respectively. The white bar has a length of 70μm. </td>
+<td>U-2 OS cells transiently transfected with <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678052">pJEJAM3</a> and <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678053">pJEJAM5</a> expressing YFP and CFP, respectively. The white bar has a length of 40μm. </td>
+</tr>
+</table>
+<br>
+<br>
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