Difference between revisions of "Part:BBa K2796002"

 
 
(One intermediate revision by the same user not shown)
Line 4: Line 4:
  
 
possibly boosts cellular metabolism by tuning up the citric acid cycle
 
possibly boosts cellular metabolism by tuning up the citric acid cycle
 +
 +
==Literature Characterization by AFCM-Egypt==
 +
The study created a reporter construct by joining the C-terminus of CD63, one of the most used exosome markers, to nanoluc (nluc), a tiny and potent bioluminescence reporter10. After progressive centrifugation to eliminate masking signals12, luminescence in the cell-culture supernatant was measured. This reporter gene was co-transfected with plasmids expressing potential candidates for exosome production augmentation.
 +
<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/booster-genes.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 study found STEAP3 syndecan-4 (SDC4), and (NadB) as potential synthetic exosome production boosters. Combined expression of these genes significantly increased exosome production, and a tricistronic plasmid vector ( known as exosome production booster), which guarantees that transfected cells receive all boosted genes at a fixed ratio ,produced a 15-fold to 40-fold increase (depending on cell conditions) in the luminescence signal in the supernatant.
 +
</span></p></div></html>
 +
==Characterization By Mutational Landscape by AFCM-Egypt==
 +
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 (H196R) shows the highest epistatic fitness, while the lowest score was associated with the mutation (C308H).
 +
<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/nadb.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 NADB.
 +
</span></p></div></html>
 +
==charactrization by mathematical modelinge by AFCM-Egypt==
 +
Presence of NadB part will aid in increasing the level of engineered exosomes so it plays an effective role to increase the efficacy of the therapeutic agent.
 +
We compared both condition of exosomes production when using booster genes and without it
 +
<br><br>
 +
(1)No booster genes with conditioned release
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:120%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 45%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/modeling/no-booster.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>
 +
<br><br>
 +
(2)Booster gene with conditioned release
 +
<html><div align="center"style="border:solid #17252A; width:100%;float:center;"><img style="                              max-width:850px;
 +
width:120%;
 +
height:auto;
 +
position: relative;
 +
top: 50%;
 +
left: 45%;
 +
transform: translate( -50%);
 +
padding-bottom:25px;
 +
padding-top:25px;
 +
"src="https://static.igem.wiki/teams/4586/wiki/modeling/booster.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>
 +
==Experimental Characterization by AFCM-Egypt==
 +
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 (P3) including NAdB, 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 (P3).
 +
<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>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 20:45, 11 October 2023


NadB

possibly boosts cellular metabolism by tuning up the citric acid cycle

Literature Characterization by AFCM-Egypt

The study created a reporter construct by joining the C-terminus of CD63, one of the most used exosome markers, to nanoluc (nluc), a tiny and potent bioluminescence reporter10. After progressive centrifugation to eliminate masking signals12, luminescence in the cell-culture supernatant was measured. This reporter gene was co-transfected with plasmids expressing potential candidates for exosome production augmentation.

The study found STEAP3 syndecan-4 (SDC4), and (NadB) as potential synthetic exosome production boosters. Combined expression of these genes significantly increased exosome production, and a tricistronic plasmid vector ( known as exosome production booster), which guarantees that transfected cells receive all boosted genes at a fixed ratio ,produced a 15-fold to 40-fold increase (depending on cell conditions) in the luminescence signal in the supernatant.

Characterization By Mutational Landscape by AFCM-Egypt

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 (H196R) shows the highest epistatic fitness, while the lowest score was associated with the mutation (C308H).

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

charactrization by mathematical modelinge by AFCM-Egypt

Presence of NadB part will aid in increasing the level of engineered exosomes so it plays an effective role to increase the efficacy of the therapeutic agent. We compared both condition of exosomes production when using booster genes and without it

(1)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.



(2)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) as the production of the engineered exosomes is initiated once the internal domain is activated.

Experimental Characterization by AFCM-Egypt

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 (P3) including NAdB, 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 (P3).

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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 406
    Illegal AgeI site found at 1026
  • 1000
    COMPATIBLE WITH RFC[1000]