Difference between revisions of "Part:BBa K1119006"
 
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<partinfo>BBa_K1119006 short</partinfo>
 
<partinfo>BBa_K1119006 short</partinfo>
  
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CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter that we used to express glyoxylate genes in mammalian cell. Our objective in characterizing this promoter is to test if the CMV promoter drives the expression of green fluorescence protein (GFP) in mammalian HEK293FT cell line.  
+
CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.
 +
<br>
 +
===Issues with Previously Submitted CMV Promoters===
 +
The Part Registry contains CMV promoter ([[Part:BBa_J52034|BBa_J52034]]) submitted by Slovenia 2006 team and [[Part:BBa_J52034:Experience#Characterisation|characterized by DTU-Denamrk 2011 team]]. However, according to the user reviews, team LMU-Munich 2010 stated that [[Part:BBa_J52034:Experience#User%20Reviews|this part is not a CMV promoter but rather a long version of ''lacI'' gene for prokaryotes]]. In addition, there are five twins of this CMV promoter, including [[Part:BBa_I712004|BBa_I712004]], which was submitted by Ljubljana 2007 team and [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Different_core_promoters_result_in_different_expression_strength| characterized by Heidelberg 2009] team using [[Part:BBa_K203100|BBa_K203100]] pSMB_MEASURE (Promoter measurement plasmid). Since we could not identify any reliable CMV promoter from the Part Registry, we decided to build one for our constitutive glyoxylate shunt construct.
 +
<br><br>
 +
<span style="color:red">Caution: If this promoter is fused to a mammalian translation unit using RFC10, the 5'UTR would only have 6nt. If users encounter lower or no expression upon assembly, including extra DNA spacer sequences between the CMV promoter and the first ATG codon might help.</span>
 +
==Literature Characterization by AFCM-Egypt==
 +
The study used Genomatix software for analysis of the CMV promoter (-550 to +48 relative to the transcription start site; TSS) for the presence of putative transcription factor regulatory elements (TFREs). They recognized one hundred and eight discrete TFREs in the CMV promoter for the presence of their cognate transcription factors (TFs) in HEK293 cells.
 +
<html><div align="center"style="border:solid #17252A; width:50%;float:center;"><img style="                              max-width:850px;
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width:75%;
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height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 35%;
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transform: translate( -50%);
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padding-bottom:25px;
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padding-top:25px;
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"src="https://static.igem.wiki/teams/4586/wiki/literature-characterisation-parts/cmv-promoter.png">
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<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'> (A) THEY MADE RNA-seq analysis of the HEK293 cell transcriptome to assess the gene expression level of TFs. Points represent the expression level of each TF sampled at exponential and stationary phases of culture. They thought that genes with more than two transcripts per million (log2 TPM > 1) are actively transcribed genes. (B) They chose a CMV promoter sequence with 25 selected TFREs for in vitro analysis. The TSS is referred to with an arrow.
 +
</span></p></div></html>
 +
==charactrizion by mathematical modeling by AFCM-Egypt==
 +
That part plays an important role for expression of the Syn-Notch receptors that bind to BCR so we used a set of ordinary differential equations (ODEs) and plotted them in order to build a model that describes the binding between the external domain (CCP1) of Syn-Notch receptor and BCR.
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:75%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 35%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/modeling/16.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'> The graph shows an increase in the free portion of CCP1 of Syn-Notch (represented as blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line).
 +
</span></p></div>
 +
</html>
 +
<br><br>
 +
Presence of the CMV part controles  booster gene expression which increases the level of engineered exosomes so it plays an effective role to increase the efficacy of the therapeutic agent.
 +
<br><br>
 +
Booster gene with conditioned release
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:75%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 35%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/modeling/pasted-image-0-1.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'> This Represents the relation between the activation of the internal domain of the Syn-Notch (represented as red line) and production of exosomes with specific cargo (represented as blue line) which increases in their level due to presence of the booster gene, Where the production of the engineered exosomes is initiated once the internal domain is activated.
 +
</span></p></div>
 +
</html>
 +
<br><br>
 +
No booster genes with conditioned release
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:75%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 35%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/modeling/pasted-image-0-2.png">
 +
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 +
lang=EN style='font-size:11.0pt;line-height:115%'> This Represents the relation between the activation of the internal domain of the Syn-Notch (represented as red line) and production of exosomes with specific cargo (represented as blue line) as the production of the engineered exosomes is initiated once the internal domain is activated.
 +
</span></p></div>
 +
</html>
  
−
The partsregistry contains CMV promoter (BBa_J52034) submitted by Slovenia 2006 team and characterized by DTU-Denamrk 2011 team. In addition, the twin of this CMV promoter BBa_I712004 was submitted by Ljubljana 2007 team and characterized by Heidelberg 2009 team using BBa_K203100 pSMB_MEASURE (Promoter measurement plasmid). However, according to the user reviews, team LMU-Munich 2010 stated that this part is not a CMV promoter but rather a long version of lacI gene for prokaryotes. Although there were many uses of this promoter, our team decided to build a reliable CMV promoter for our constitutive glyoxylate shunt construct.
 
  
 +
===Characterization===
 +
In our characterization, CMV promoter was assembled with GFP reporter ([[Part:BBa_K648013|BBa_K648013]]) and hGH polyA terminator ([[Part:BBa_K404108|BBa_K404108]]).
 +
 +
The pCMV-GFP was then transfected into HEK293FT cells and <i>in vivo</i> green fluorescence signal was observed under confocal microscope.
 +
 +
The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator ([[Part:BBa_K648013|BBa_K648013]]) that does not contain the CMV promoter.
 +
 +
The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization/cmv detailed protocol] of our characterization can be found in HKUST iGEM 2013 Wiki.
 +
 +
[[File:Final CMV annotated no ABC.jpg|500px|thumb|center|'''Figure 1. CMV promoter drives expression of GFP.''' HEK cells transfected with pCMV-GFP gave GFP signals. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals. Scale bar = 10 microns]]
 +
 +
==Egypt-AFCM Team Improvement==
 +
 +
[http://2017.igem.org/Team:AFCM-Egypt# Egypt-AFCM Team] tried to improve this part by Fusing CMV enhancer to CMV promoter in one composite part to enhance its transcription activity and was resubmitted at [https://parts.igem.org/wiki/index.php?title=Part:BBa_K2217024# BBa_K2217024]. Part characterization and usage can be found at [https://parts.igem.org/Part:BBa_K2217024:Experience# Part Experience]
 +
 +
==MIT-2019 Characterization==
 +
 +
While the majority of our project was focused on engineering leader cells, we were also interested in manipulating the follower cells by genetically engineering HL-60 cells. We noticed that while there were several methods of transfection described for HL-60 cells ( including a project by the <a href=”http://2009.igem.org/Team:UCSF”>2009 UCSF iGEM team</a>, a paper by <a href=”http://limlab.ucsf.edu/papers/pdfs/park_2014.pdf”>Park et. al.</a>), we did not find any systematic data on the function of commonly used promoters in this cell type. Considering that HL-60 cells are relatively difficult to transfect and require harsh transfection conditions (electroporation) that can result in cell death and low transfection efficiency, we wanted to find a promoter that would lead to reliable and strong expression of transfected genes in order to facilitate our future experiments with the SynNotch system and engineering of leader cells to become followers.
 +
 +
In particular, we characterized the expression of the fluorescent proteins EYFP and TagBFP encoded on plasmids under the CMV and hEF1a promoters and transfected by electroporation into undifferentiated HL-60 cells.
 +
 +
Figure 1:
 +
https://static.igem.org/mediawiki/parts/5/54/T--MIT--PartsFigure1.png
 +
 +
Figure 2a:
 +
https://static.igem.org/mediawiki/parts/3/38/T--MIT----PartsFigure2a.png
 +
 +
Figure 2b:
 +
https://static.igem.org/mediawiki/parts/3/33/T--MIT----PartsFigure2b.png
 +
 +
We observed a lot of cell death due to electroporation. Events from the flow cytometry analysis were first plotted on an FSC/SSC dot-plot graph to set an analysis gate, as shown in Figure 1. For the cells within the analyzed gate we looked at fluorescence in the FITC channel (excitation 488 nm, detection window 530/30 nm) for detection of EYFP and Pacific Blue channel (excitation 405 nm, detection window 450/50 nm) for detection of TagBFP. We found that 52% of cells transfected with CMV-EYFP were fluorescent in the yellow channel, and 35% of cells transfected with CMV-TagBFP were fluorescent in the blue channel. On the other hand, only 1.5% of cells transfected with hEF1a-EYFP and 8% of cells transfected with hEF1A-TagBFP were weakly fluorescent.
 +
 +
Figure 2 shows the overlay of histograms for untransfected cells (control, shown in green) and cells transfected with the fluorescent protein encoded under a CMV promoter (shown in blue) or hEF1a promoter (shown in red) for a) TagBFP and b) EYFP. 
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===
 +
 +
  
 
<!-- -->
 
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Latest revision as of 19:46, 11 October 2023

CMV promoter

CMV (Cytomegalovirus) promoter is a constitutive mammalian promoter.

Issues with Previously Submitted CMV Promoters

The Part Registry contains CMV promoter (BBa_J52034) submitted by Slovenia 2006 team and characterized by DTU-Denamrk 2011 team. However, according to the user reviews, team LMU-Munich 2010 stated that this part is not a CMV promoter but rather a long version of lacI gene for prokaryotes. In addition, there are five twins of this CMV promoter, including BBa_I712004, which was submitted by Ljubljana 2007 team and [http://2009.igem.org/Team:Heidelberg/Project_Measurement#Different_core_promoters_result_in_different_expression_strength| characterized by Heidelberg 2009] team using BBa_K203100 pSMB_MEASURE (Promoter measurement plasmid). Since we could not identify any reliable CMV promoter from the Part Registry, we decided to build one for our constitutive glyoxylate shunt construct.

Caution: If this promoter is fused to a mammalian translation unit using RFC10, the 5'UTR would only have 6nt. If users encounter lower or no expression upon assembly, including extra DNA spacer sequences between the CMV promoter and the first ATG codon might help.

Literature Characterization by AFCM-Egypt

The study used Genomatix software for analysis of the CMV promoter (-550 to +48 relative to the transcription start site; TSS) for the presence of putative transcription factor regulatory elements (TFREs). They recognized one hundred and eight discrete TFREs in the CMV promoter for the presence of their cognate transcription factors (TFs) in HEK293 cells.

(A) THEY MADE RNA-seq analysis of the HEK293 cell transcriptome to assess the gene expression level of TFs. Points represent the expression level of each TF sampled at exponential and stationary phases of culture. They thought that genes with more than two transcripts per million (log2 TPM > 1) are actively transcribed genes. (B) They chose a CMV promoter sequence with 25 selected TFREs for in vitro analysis. The TSS is referred to with an arrow.

charactrizion by mathematical modeling by AFCM-Egypt

That part plays an important role for expression of the Syn-Notch receptors that bind to BCR so we used a set of ordinary differential equations (ODEs) and plotted them in order to build a model that describes the binding between the external domain (CCP1) of Syn-Notch receptor and BCR.

The graph shows an increase in the free portion of CCP1 of Syn-Notch (represented as blue line) then decreases as BCR binds to it. As the binding occurs, BCR decreases (represented as orange line) and the binding state increases (represented as green line).



Presence of the CMV part controles booster gene expression which increases the level of engineered exosomes so it plays an effective role to increase the efficacy of the therapeutic agent.

Booster gene with conditioned release

This Represents the relation between the activation of the internal domain of the Syn-Notch (represented as red line) and production of exosomes with specific cargo (represented as blue line) which increases in their level due to presence of the booster gene, Where the production of the engineered exosomes is initiated once the internal domain is activated.



No booster genes with conditioned release

This Represents the relation between the activation of the internal domain of the Syn-Notch (represented as red line) and production of exosomes with specific cargo (represented as blue line) as the production of the engineered exosomes is initiated once the internal domain is activated.


Characterization

In our characterization, CMV promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108).

The pCMV-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under confocal microscope.

The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. A negative control was made by GFP generator (BBa_K648013) that does not contain the CMV promoter.

The [http://2013.igem.org/Team:Hong_Kong_HKUST/characterization/cmv detailed protocol] of our characterization can be found in HKUST iGEM 2013 Wiki.

Figure 1. CMV promoter drives expression of GFP. HEK cells transfected with pCMV-GFP gave GFP signals. HEK cells transfected with the commercial pEGFP-N1 showed similar results, while the same construct without any promoter did not give any GFP signals. Scale bar = 10 microns

Egypt-AFCM Team Improvement

[http://2017.igem.org/Team:AFCM-Egypt# Egypt-AFCM Team] tried to improve this part by Fusing CMV enhancer to CMV promoter in one composite part to enhance its transcription activity and was resubmitted at BBa_K2217024. Part characterization and usage can be found at Part Experience

MIT-2019 Characterization

While the majority of our project was focused on engineering leader cells, we were also interested in manipulating the follower cells by genetically engineering HL-60 cells. We noticed that while there were several methods of transfection described for HL-60 cells ( including a project by the <a href=”http://2009.igem.org/Team:UCSF”>2009 UCSF iGEM team</a>, a paper by <a href=”http://limlab.ucsf.edu/papers/pdfs/park_2014.pdf”>Park et. al.</a>), we did not find any systematic data on the function of commonly used promoters in this cell type. Considering that HL-60 cells are relatively difficult to transfect and require harsh transfection conditions (electroporation) that can result in cell death and low transfection efficiency, we wanted to find a promoter that would lead to reliable and strong expression of transfected genes in order to facilitate our future experiments with the SynNotch system and engineering of leader cells to become followers.

In particular, we characterized the expression of the fluorescent proteins EYFP and TagBFP encoded on plasmids under the CMV and hEF1a promoters and transfected by electroporation into undifferentiated HL-60 cells.

Figure 1: T--MIT--PartsFigure1.png

Figure 2a: T--MIT----PartsFigure2a.png

Figure 2b: T--MIT----PartsFigure2b.png

We observed a lot of cell death due to electroporation. Events from the flow cytometry analysis were first plotted on an FSC/SSC dot-plot graph to set an analysis gate, as shown in Figure 1. For the cells within the analyzed gate we looked at fluorescence in the FITC channel (excitation 488 nm, detection window 530/30 nm) for detection of EYFP and Pacific Blue channel (excitation 405 nm, detection window 450/50 nm) for detection of TagBFP. We found that 52% of cells transfected with CMV-EYFP were fluorescent in the yellow channel, and 35% of cells transfected with CMV-TagBFP were fluorescent in the blue channel. On the other hand, only 1.5% of cells transfected with hEF1a-EYFP and 8% of cells transfected with hEF1A-TagBFP were weakly fluorescent.

Figure 2 shows the overlay of histograms for untransfected cells (control, shown in green) and cells transfected with the fluorescent protein encoded under a CMV promoter (shown in blue) or hEF1a promoter (shown in red) for a) TagBFP and b) EYFP. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]