Difference between revisions of "Part:BBa K4586013"
(→‎Experimental Characterization)
 
(7 intermediate revisions by 3 users not shown)
Line 4: Line 4:
  
 
==Part Description==
 
==Part Description==
−
It is a transmembrane protein called connexin (CX43) that facilitates intracellular communication.
+
This part is a transmembrane protein called connexin (CX43) that oligomerizes to form hexameric channels, called hemichannels, that can control the interaction and transfer of information between exosomes and cells of interest, which is called intercellular communication.
 +
 
 
==Usage==
 
==Usage==
  
 
This part is implicated in our design to increase the amount of our therapeutic agent within the exosomes, as the CX43 transmembrane portion facilitates the loading of our cargo into the exosomes.
 
This part is implicated in our design to increase the amount of our therapeutic agent within the exosomes, as the CX43 transmembrane portion facilitates the loading of our cargo into the exosomes.
 +
==Literature Characterization==
 +
The study made western blot analyses of extracts from various cell lines to find out the effect of connexin 43 in different cells.
 +
<html><div align="center"style="border:solid #17252A; width:50%;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/literature-characterisation-parts/cx43.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%'>By using Connexin 43 (E7N2R) XP Rabbit mAb in the upper and GAPDH (D16H11) XP Rabbit mAb in the lower. Connexin 43 protein is expressed at low levels in BeWo cells, which is compatible with the expected expression pattern.
 +
</span></p></div></html>
 +
<br><br><br><br>
 +
<html><div align="center"style="border:solid #17252A; width:50%;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/literature-characterisation-parts/3.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%'>In lane 1, we have Western blot analysis of extracts from control 293T cells; in lane 2, we have Western blot analysis of connexin 43 knockout 293T cells, and that was done using Connexin 43  XP Rabbit mAb in the upper and GAPDH  XP Rabbit mAb  in the lower.
 +
Connexin 43 antibody specificity is demonstrated by the absence of a signal in connexin 43 knockout 293T cells.
 +
 +
</span></p></div></html>
 +
<br><br>
 +
==Characterization By Mutational Landscape==
 +
In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness.  As displayed in the chart below, the mutation (V236I) shows the highest epistatic fitness, while the lowest score was associated with the mutation (K109Q,S244R).
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-de/cx43.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%'>Figure . An illustration of the effects of different mutations on the Epistatic Fitness of CX43.
 +
</span></p></div></html>
 +
==Experimental Characterization==
 +
In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P6) including CX43, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/pcr-ampli.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%'>
 +
 +
</span></p></div></html>
 +
<br><br><br><br>
 +
We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P6).
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/parts-experiments/digestion-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%'>
 +
 +
</span></p></div></html>
 +
<br><br>
 +
After the ligation step, we did a culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. This figure shows the Cell culture plate of transformed pCDNA vector 2 containing insert parts.
 +
This plasmid contains Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43)
 +
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 +
width:100%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 50%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/results/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%'>
 +
 +
</span></p></div></html>
 +
 +
==References==
 +
Giepmans, B.N. et al. (2001) J Biol Chem 276, 8544-9.
 +
<br><br>
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 14:02, 12 October 2023


CX43

Part Description

This part is a transmembrane protein called connexin (CX43) that oligomerizes to form hexameric channels, called hemichannels, that can control the interaction and transfer of information between exosomes and cells of interest, which is called intercellular communication.

Usage

This part is implicated in our design to increase the amount of our therapeutic agent within the exosomes, as the CX43 transmembrane portion facilitates the loading of our cargo into the exosomes.

Literature Characterization

The study made western blot analyses of extracts from various cell lines to find out the effect of connexin 43 in different cells.

By using Connexin 43 (E7N2R) XP Rabbit mAb in the upper and GAPDH (D16H11) XP Rabbit mAb in the lower. Connexin 43 protein is expressed at low levels in BeWo cells, which is compatible with the expected expression pattern.





In lane 1, we have Western blot analysis of extracts from control 293T cells; in lane 2, we have Western blot analysis of connexin 43 knockout 293T cells, and that was done using Connexin 43 XP Rabbit mAb in the upper and GAPDH XP Rabbit mAb in the lower. Connexin 43 antibody specificity is demonstrated by the absence of a signal in connexin 43 knockout 293T cells.



Characterization By Mutational Landscape

In order to optimize the function of our parts, we've used the concept of Directed Evolution through applying different mutations and measuring the effects of these mutations on their evolutionary epistatic fitness. As displayed in the chart below, the mutation (V236I) shows the highest epistatic fitness, while the lowest score was associated with the mutation (K109Q,S244R).

Figure . An illustration of the effects of different mutations on the Epistatic Fitness of CX43.

Experimental Characterization

In order to amplify this DNA part, we used PCR amplification to reach the desired concentration to complete our experiments using specific forward and reverse primers, running the parts on gel electrophoresis as this part presents in lane (P6) including CX43, and then measuring the specific concentration of the running part using Real-Time PCR as shown in the following figure.





We performed the double digestion method for this part in the prefix and suffix with its specific restriction enzyme and applied this part to gel electrophoresis as shown in the following figure lane (P6).



After the ligation step, we did a culture of the ligated product to specifically select the optimum colonies to screen it using Colony PCR to make sure that our parts were correctly ligated in the plasmid. This figure shows the Cell culture plate of transformed pCDNA vector 2 containing insert parts. This plasmid contains Loading system(CD63-L7Ae)-exosomal receptor(LAMP2B-anti-CD19)-connexin(CX43)

References

Giepmans, B.N. et al. (2001) J Biol Chem 276, 8544-9.

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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 782