Difference between revisions of "Part:BBa K4586001"

(→‎charactrization of mathematical modeling)
(→‎Experimental Characterization)
 
(8 intermediate revisions by 2 users not shown)
Line 52: Line 52:
 
  </span></p></div></html>
 
  </span></p></div></html>
 
==charactrization of mathematical modeling==
 
==charactrization of mathematical modeling==
−
This model is based on an assumption that the external domain activation will lead to transmembrane domain activation of our synthetic notch receptor which starts from cell to cell interaction till activation of the internal domain ZF21.16VP64.
+
This model is based on an assumption that the external domain activation will lead to transmembrane domain activation of our Syn-Notch receptor which starts from cell to cell interaction till activation of the internal domain ZF21.16VP64.
 
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 
<html><div align="center"style="border:solid #17252A; width:80%;float:center;"><img style="                              max-width:850px;
 
width:100%;
 
width:100%;
Line 62: Line 62:
 
padding-bottom:25px;
 
padding-bottom:25px;
 
padding-top:25px;
 
padding-top:25px;
−
"src="https://static.igem.wiki/teams/4586/wiki/modeling/pasted-image-0.png">
+
"src="https://static.igem.wiki/teams/4586/wiki/modeling/pasted-image-1.png">
 
<p class=MsoNormal align=center style='text-align:left;border:none;width:98% ;justify-content:center;'><span
 
<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%'>As the binding state occurs, It is evident that the signal is transmitted through the transmembrane domain(mouth notch core) that is resulted from cell to cell interaction..  
 
lang=EN style='font-size:11.0pt;line-height:115%'>As the binding state occurs, It is evident that the signal is transmitted through the transmembrane domain(mouth notch core) that is resulted from cell to cell interaction..  
 +
</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 (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and anti CD19 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 in lane (P1).
 +
<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 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.
 +
The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure.
 +
This plasmid contains
 +
1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64))
 +
2-Booster gene 1 (SDC4, STEAP3)
 +
3-Booster gene 2 (NAdB)
 +
<br>
 +
<html><div align="center"style="border:solid #17252A; width:75%;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/parts/vector-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%'>
 +
 
  </span></p></div></html>
 
  </span></p></div></html>
  

Latest revision as of 14:01, 12 October 2023


Mouse notch core

Part Description

This part codes for an enhanced Mouse notch core, which is a notch-derived transmembrane regulatory protein that normally regulates cell fate decisions during a variety of developmental processes. It was first identified in beaded winged flies. The activity of the synthetic notch is determined by the sequential cleavage of its domain carried out by protease enzymes after cell interaction with another cell.


Usage

Mouse notch is implicated in our design as the transmembrane domain of our Syn notch receptor as it transmits the signal that is started by cell-to-cell contact between the external domain and the autoreactive B cells through it’s mechanosensitive quality based on proteolytic cleavage of its domains to release our intra-cellular transcription module (ZF21.16 VP64) that activates the ZF21.16 minCMV promoter controlling the expression of our cargo as shown in figure 1.

Figure 1. shows how the mouse notch core protein within the syn notch receptor is expressed as a transmembrane domain to transmit the signal initiated by interaction between the autoreactive B-cell receptor and our engineered MSC

Literature Characterization

The study used X-Gal staining of brain sections from double transgenic mice to show the effect of active Notch1 signaling in mice. The results suggest that the activation of the N1-Gal4VP16 reporter is consistent with the activity of the endogenous Notch1 receptor. This reporter mouse is a powerful tool for visualizing active Notch1 signaling in embryonic and post-natal development in vivo.

The N1-Gal4VP16 activity in BAC-transgenic reporter mice correlates well with the expected profile of active Notch1 signaling. This is evident from the X-Gal staining of brain sections from double transgenic mice, which shows strong staining in the left lateral ventricle (LV), a region of high Notch1 activity. Immunohistochemistry staining with cleaved Notch1 (Val1744) antibody confirms this finding.

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 (V67E) shows the highest epistatic fitness, while the lowest score was associated with the mutation (S312R).

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

charactrization of mathematical modeling

This model is based on an assumption that the external domain activation will lead to transmembrane domain activation of our Syn-Notch receptor which starts from cell to cell interaction till activation of the internal domain ZF21.16VP64.

As the binding state occurs, It is evident that the signal is transmitted through the transmembrane domain(mouth notch core) that is resulted from cell to cell interaction..

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 (P1) including CD8 alpha-his tag-mouse notch core-ZF21.16\VP64, and anti CD19 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 in lane (P1).



After the ligation step, we did 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. The cell culture plate of transformed pCDNA vector containing insert parts is shown in the following figure. This plasmid contains 1-Syn-notch (CD8 alpha-his tag-Anti CD19-mouse notch core-ZF21.16\VP64)) 2-Booster gene 1 (SDC4, STEAP3) 3-Booster gene 2 (NAdB)

References

Generation and characterization of a Notch1 signaling‐specific reporter mouse line - Smith - 2012 - genesis - Wiley Online Library

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 SapI.rc site found at 592