Difference between revisions of "Part:BBa K2065003"
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2065003 short</partinfo>
 
<partinfo>BBa_K2065003 short</partinfo>
  
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This BioBrick is a coding sequence for the CT52 protein, attached to the Caspase 9 protein with a GGS10 linker in between. Because CT52 has a binding interaction for the T14-3-3 scaffold protein under influence of the small molecule fusicoccin, it can be used for assembly of proteins on this scaffold protein.  
+
Caspase9-CT52 This BioBrick is a coding sequence for the CT52 protein, attached to the Caspase 9 protein with a GGS10 linker in between. Because CT52 has a binding interaction for the T14-3-3 scaffold protein under influence of the small molecule fusicoccin, it can be used for assembly of proteins on this scaffold protein.
  
−
Design of the construct
 
−
This parts is constructed of three subparts; CT52, a GGS10 linker and Caspase 9. It has a molecular weight of 52 kDa. CT52 is the name for the isolated last 52 C-terminal amino acids of the plant plasma H+-ATPase PMA2 [1]. The N-terminus of CT52 is connected to a flexible GGS10 linker. This linker consists of a ten times repeating Glycine-Glycine-Serine amino acid sequence.
 
  
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Usage
+
===Design of the construct===
−
Caspase 9 is an apoptosis inducer which can activates by dimerizing with itself. The ability of caspase 9 to cleave the Ac-LEHD-AFC substrate can be used to determine the caspase 9 activity. When two caspase-9 proteins dimerize, and cleave Ac-LEHD-AFC, this substrate splits into a non-fluorescent peptide and a fluorescent part. This fluorescence has an excitation of 400 nm and emission  at 505 nm. Thus caspase 9 activity can be measured by means of fluorescence. [1]
+
This parts is constructed of three subparts; CT52, a GGS10 linker and Caspase 9. It has a molecular weight of 52 kDa. CT52 is the name for the isolated last 52 C-terminal amino acids of the plant plasma H+-ATPase PMA2 [1]. The N-terminus of CT52 is connected to a flexible GGS10 linker. This linker consists of a ten times repeating Glycine-Glycine-Serine amino acid sequence.
−
When caspase-9 and CT52 are linked, the binding affinity of CT52 and the T14-3-3 construct can be measured by means of fluorescence . Because caspase 9 is an apoptosis inducer so Caspase9-CT52 could also be used in a T14-3-3 based kill switch.
+
  
−
iGEM TU Eindhoven used this part to characterize a tetrameric variant of the T14-3-3 scaffold protein. Unfortunately constructing this protein did not succeed, so no experimental data was obtained from this protein.
 
  
−
Sequence
+
===Usage===
 +
Caspase 9 is an apoptosis inducer which can activates by dimerizing with itself. The ability of caspase 9 to cleave the Ac-LEHD-AFC substrate can be used to determine the caspase 9 activity. When two caspase-9 proteins dimerize, and cleave Ac-LEHD-AFC, this substrate splits into a non-fluorescent peptide and a fluorescent part. This fluorescence has an excitation of 400 nm and emission  at 505 nm. Thus caspase 9 activity can be measured by means of fluorescence. [1]
 +
When caspase-9 and CT52 are linked, the binding affinity of CT52 and the T14-3-3 construct can be measured by means of fluorescencei. Because caspase 9 is an apoptosis inducer so Caspase9-CT52 could also be used in a T14-3-3 based kill switch.
 +
===Sequence===
 
The sequence of our Capase9-CT52 has been verified by StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). Caspase9-CT52 is 1386 bp long.
 
The sequence of our Capase9-CT52 has been verified by StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). Caspase9-CT52 is 1386 bp long.
  
 +
===Sequence===
 +
The sequence of our Capase9-CT52 has been verified by StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). Caspase9-CT52 is 1386 bp long. The specifications of the protein can be found below.
  
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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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−
<!-- -->
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2065003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2065003 SequenceAndFeatures</partinfo>
  
 +
===Characterization===
 +
iGEM TU Eindhoven planned to use this part to characterize a tetrameric variant of the T14-3-3 scaffold protein. The constructing of this protein succeeded on DNA level, but due to time limitations the protein couldn't be expressed. To show the caspase 9 dimerization this was tested on a homodimeric T14-3-3 scaffold protein. 
 +
 +
 +
===Protein Specifications===
 +
 +
<table>
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<tr>
 +
<th style="height:40px;" colspan="4"> Protein Specifications</th>
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</tr>
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<tr>
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<td style="font-weight:bold;" rowspan="6">General Information</td>
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<td colspan="2">Number of amino acids</td>
 +
<td> 475</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Molecular weight</td>
 +
<td> 51624</td>
 +
</tr>
 +
<tr>
 +
<td colspan="2">Theoretical pi</td>
 +
<td> 5.01</td>
 +
</tr>
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<tr>
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<td colspan="2">Extinction coefficient</td>
 +
<td> 27555</td>
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</tr>
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<tr>
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<td colspan="2">Formula</td>
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<td> C<sub>2248</sub>H<sub>3538</sub>N<sub>626</sub>O<sub>730</sub>S<sub>19</sub></td>
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</tr>
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<tr>
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<td colspan="2">Total numbers of atons</td>
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<td> 7161 </td>
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</tr>
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<tr>
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<td style="font-weight:bold;" rowspan="40">Amino Acid Composition</td>
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<td style="font-weight:bold;">Amino Acid</td>
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<td style="font-weight:bold;">Frequency</td>
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<td style="font-weight:bold;">Percentage(%)</td>
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</tr>
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<tr>
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<td>Ala(A)</td>
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<td>26</td>
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<td>5.5</td>
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</tr>
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<tr>
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<td>Arg(R</td>
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<td>12</td>
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<td>4.4</td>
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</tr>
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<tr>
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<td>Asn(N)</td>
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<td>16</td>
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<td>3.4</td>
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</tr>
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<tr>
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<td>Asp(D)</td>
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<td>27</td>
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<td>5.7</td>
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</tr>
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<tr>
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<td>Cys(C)</td>
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<td>11</td>
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<td>2.3</td>
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</tr>
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<tr>
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<td>Gln(Q)</td>
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<td>20</td>
 +
<td>4.2</td>
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</tr>
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<tr>
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<td>Glu(E)</td>
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<td>43</td>
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<td>9.1</td>
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</tr>
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<tr>
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<td>Gly(G)</td>
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<td>52</td>
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<td>10.9</td>
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</tr>
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<tr>
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<td>His(H)</td>
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<td>11</td>
 +
<td>2.3</td>
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</tr>
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<tr>
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<td>Ile(I)</td>
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<td>24</td>
 +
<td>5.1</td>
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</tr>
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<tr>
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<td>Leu(L)</td>
 +
<td>40</td>
 +
<td>8.4</td>
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</tr>
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<tr>
 +
<td>Lys(K)</td>
 +
<td>27</td>
 +
<td>5.7</td>
 +
</tr>
 +
<tr>
 +
<td>Met(M)</td>
 +
<td>8</td>
 +
<td>1.7</td>
 +
</tr>
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<tr>
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<td>Phe(F)</td>
 +
<td>23</td>
 +
<td>4.8</td>
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</tr>
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<tr>
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<td>Pro(P)</td>
 +
<td>20</td>
 +
<td>4.2</td>
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</tr>
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<tr>
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<td>Ser(S)</td>
 +
<td>44</td>
 +
<td>9.3</td>
 +
</tr>
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<tr>
 +
<td>Thr(T)</td>
 +
<td>27</td>
 +
<td>5.7</td>
 +
</tr>
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<tr>
 +
<td>Trp(W)</td>
 +
<td>3</td>
 +
<td>0.6</td>
 +
</tr>
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<tr>
 +
<td>Tyr(T)</td>
 +
<td>7</td>
 +
<td>1.5</td>
 +
</tr>
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<tr>
 +
<td>Val(V)</td>
 +
<td>25</td>
 +
<td>5.3</td>
 +
</tr>
 +
<tr>
 +
<td>Pyl(O)</td>
 +
<td>0</td>
 +
<td>0.0</td>
 +
</tr>
 +
<tr>
 +
<td>Sec(U)</td>
 +
<td>0</td>
 +
<td>0.0</td>
 +
</tr>
 +
</table>
 +
 +
 +
===References===
 +
[1] Pop, C., Timmer, J., Sperandio, S., & Salvesen, G. (2006). The Apoptosome Activates Caspase-9 by Dimerization. Molecular Cell, 22(2), 269-275. 
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Revision as of 22:15, 15 October 2016

CT52-Caspase9

Caspase9-CT52 This BioBrick is a coding sequence for the CT52 protein, attached to the Caspase 9 protein with a GGS10 linker in between. Because CT52 has a binding interaction for the T14-3-3 scaffold protein under influence of the small molecule fusicoccin, it can be used for assembly of proteins on this scaffold protein.


Design of the construct

This parts is constructed of three subparts; CT52, a GGS10 linker and Caspase 9. It has a molecular weight of 52 kDa. CT52 is the name for the isolated last 52 C-terminal amino acids of the plant plasma H+-ATPase PMA2 [1]. The N-terminus of CT52 is connected to a flexible GGS10 linker. This linker consists of a ten times repeating Glycine-Glycine-Serine amino acid sequence.


Usage

Caspase 9 is an apoptosis inducer which can activates by dimerizing with itself. The ability of caspase 9 to cleave the Ac-LEHD-AFC substrate can be used to determine the caspase 9 activity. When two caspase-9 proteins dimerize, and cleave Ac-LEHD-AFC, this substrate splits into a non-fluorescent peptide and a fluorescent part. This fluorescence has an excitation of 400 nm and emission at 505 nm. Thus caspase 9 activity can be measured by means of fluorescence. [1] When caspase-9 and CT52 are linked, the binding affinity of CT52 and the T14-3-3 construct can be measured by means of fluorescencei. Because caspase 9 is an apoptosis inducer so Caspase9-CT52 could also be used in a T14-3-3 based kill switch.

Sequence

The sequence of our Capase9-CT52 has been verified by StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). Caspase9-CT52 is 1386 bp long.

Sequence

The sequence of our Capase9-CT52 has been verified by StarSeq. It contains the prefix and suffix with the correct restriction sites (EcoRI, XbaI, SpeI and PstI). Caspase9-CT52 is 1386 bp long. The specifications of the protein can be found below.

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]

Characterization

iGEM TU Eindhoven planned to use this part to characterize a tetrameric variant of the T14-3-3 scaffold protein. The constructing of this protein succeeded on DNA level, but due to time limitations the protein couldn't be expressed. To show the caspase 9 dimerization this was tested on a homodimeric T14-3-3 scaffold protein.


Protein Specifications

Protein Specifications
General Information Number of amino acids 475
Molecular weight 51624
Theoretical pi 5.01
Extinction coefficient 27555
Formula C2248H3538N626O730S19
Total numbers of atons 7161
Amino Acid Composition Amino Acid Frequency Percentage(%)
Ala(A) 26 5.5
Arg(R 12 4.4
Asn(N) 16 3.4
Asp(D) 27 5.7
Cys(C) 11 2.3
Gln(Q) 20 4.2
Glu(E) 43 9.1
Gly(G) 52 10.9
His(H) 11 2.3
Ile(I) 24 5.1
Leu(L) 40 8.4
Lys(K) 27 5.7
Met(M) 8 1.7
Phe(F) 23 4.8
Pro(P) 20 4.2
Ser(S) 44 9.3
Thr(T) 27 5.7
Trp(W) 3 0.6
Tyr(T) 7 1.5
Val(V) 25 5.3
Pyl(O) 0 0.0
Sec(U) 0 0.0


References

[1] Pop, C., Timmer, J., Sperandio, S., & Salvesen, G. (2006). The Apoptosome Activates Caspase-9 by Dimerization. Molecular Cell, 22(2), 269-275.