Difference between revisions of "Part:BBa K3503004"

 
 
(5 intermediate revisions by 2 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K3503004 short</partinfo>
 
<partinfo>BBa_K3503004 short</partinfo>
  
−
CBD-SR
+
This is a composite part designed to improve the fire retardancy with cellulose-binding domain K1321014. The protein sequence in this part is based on the K1608000 sequence from MingDao 2015 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 17: Line 17:
 
<partinfo>BBa_K3503004 parameters</partinfo>
 
<partinfo>BBa_K3503004 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
 +
<html>
 +
<style>
 +
.legend {
 +
  font-size: .9em;
 +
}
 +
p {
 +
  text-align: justify;
 +
}
 +
.content img {
 +
  max-height:80vh;
 +
  max-width:100%;
 +
  margin: 0 auto;
 +
  clear: both;
 +
}
 +
* {
 +
  box-sizing: border-box;
 +
}
 +
.column1 {
 +
  float: left;
 +
  width: 50%;
 +
  padding: 5px;
 +
}
 +
 +
/* Clearfix (clear floats) */
 +
.row::after {
 +
  content: "";
 +
  clear: both;
 +
  display: table;
 +
}
 +
</style>
 +
<div class="content">
 +
<h2>Sequencing and sequence alignment of K3503004</h2>
 +
 +
<img src="https://2020.igem.org/wiki/images/1/1c/T--Puiching_Macau--CBD_sequence.png">
 +
<div class="legend">Figure 1. Representative sequencing results of K3503004
 +
 +
<p>To future confirm that our constructs have the correct sequence, we send our clones (with positive PCR results) out to sequencing.
 +
<br>
 +
The sequencing results show that we have successfully assembled target genes K3503004  to the vector (pET-11a).
 +
 +
<h2>Successful expression of K3503004</h2>
 +
<p>In order to validate the protein expression of K1608000, K3503006 and K3503004, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make K3503001, K3503007 and K3503008 easily detectable via Western Blot, we designed our constructs to include a “His tag” which is targeted by a commercially available antibody. With the SDS-PAGE and western blot (anti-his for his-tagged protein), we found successful expression of K1608000, K3503006 and K3503004<br>
 +
Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K1608000, K3503006 and K3503004 under control of the T7 promoter. Cells were induced with0.1mM IPTG until either OD0.4 (wavelength: 600nm) at 30°C. After expression, cells were then lysed according to our protein extraction protocol, and supernatant and pellet were collected for the SDS-PAGE.
 +
</p>
 +
For more experimental details of the Western Blot and SDS-PAGE, please see <a href="https://2020.igem.org/Team:PuiChing_Macau/Protocol">protocol</a>.
 +
 +
<div class="fig">
 +
<div class="row">
 +
  <div class="column1">
 +
    <img src="https://2020.igem.org/wiki/images/9/9b/T--Puiching_Macau--SR_Blue.png" style="width:100%">
 +
    <p style="text-align: center">(a) Coomassie blue staining</p>
 +
  </div>
 +
  <div class="column1">
 +
    <img src="https://2020.igem.org/wiki/images/8/85/T--Puiching_Macau--SR_western.png" style="width:100%">
 +
    <p style="text-align: center">(b) Western Blot</p>
 +
  </div>
 +
</div>
 +
<div class="legend">Figure 2. Western Blot results of inducible expression of K1608000, K3503006, K3503004
 +
<br>
 +
<ul>
 +
<li>Lane1: Bio rad protein dual color ladder;
 +
<li>Lane2: pet11a</li>
 +
<li>Lane3: SR(K1608000)</li>
 +
<li>Lane4:SR(K1608000) with 1mM IPTG induction</li>
 +
<li>Lane5: K3503006(mfp5-SR)</li>
 +
<li>Lane6: K3503006(mfp5-SR) with 1mM IPTG induction</li>
 +
<li>Lane7: K3503004(CBD-SR)</li>
 +
<li>Lane8: K3503004(CBD-SR) with 1mM IPTG induction</li>
 +
</ul>
 +
</div>
 +
 +
<h2>Fire retardant test of K3503004</h2>
 +
<p>The test subject was in the size of bed sheet in 300*130mm and the average Moisture content% is 13.55%-14.37%. As demonstrated on Fig 3, all of our engineered flame retardant displayed an improvement compared with the water control.</p>
 +
<img src="https://2020.igem.org/wiki/images/9/9c/T--Puiching_Macau--bedsheet_chart.png">
 +
<div class="legend">Figure 3. Vertical Burning Test using bedsheet.   
 +
<img src="https://2020.igem.org/wiki/images/2/2e/T--Puiching_Macau--wood.png">
 +
<div class="legend">Figure 4. Flame retardancy test using wood.
 +
<p>As demonstrated in Fig 4, all of our engineered K3503001, K3503007 and K3507008 flame retardants displayed an improvement compared with the water control.  </p>
 +
 +
</html>

Latest revision as of 21:57, 27 October 2020


CBD-SR-His

This is a composite part designed to improve the fire retardancy with cellulose-binding domain K1321014. The protein sequence in this part is based on the K1608000 sequence from MingDao 2015

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 146
    Illegal BglII site found at 263
    Illegal BglII site found at 332
    Illegal BamHI site found at 107
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Sequencing and sequence alignment of K3503004

Figure 1. Representative sequencing results of K3503004

To future confirm that our constructs have the correct sequence, we send our clones (with positive PCR results) out to sequencing.
The sequencing results show that we have successfully assembled target genes K3503004 to the vector (pET-11a).

Successful expression of K3503004

In order to validate the protein expression of K1608000, K3503006 and K3503004, we performed SDS-PAGE (coomassie blue staining) and Western blot analysis. To make K3503001, K3503007 and K3503008 easily detectable via Western Blot, we designed our constructs to include a “His tag” which is targeted by a commercially available antibody. With the SDS-PAGE and western blot (anti-his for his-tagged protein), we found successful expression of K1608000, K3503006 and K3503004
Before performing the SDS-PAGE, we grew up our engineered E. coli cells containing a plasmid with K1608000, K3503006 and K3503004 under control of the T7 promoter. Cells were induced with0.1mM IPTG until either OD0.4 (wavelength: 600nm) at 30°C. After expression, cells were then lysed according to our protein extraction protocol, and supernatant and pellet were collected for the SDS-PAGE.

For more experimental details of the Western Blot and SDS-PAGE, please see protocol.

(a) Coomassie blue staining

(b) Western Blot

Figure 2. Western Blot results of inducible expression of K1608000, K3503006, K3503004
  • Lane1: Bio rad protein dual color ladder;
  • Lane2: pet11a
  • Lane3: SR(K1608000)
  • Lane4:SR(K1608000) with 1mM IPTG induction
  • Lane5: K3503006(mfp5-SR)
  • Lane6: K3503006(mfp5-SR) with 1mM IPTG induction
  • Lane7: K3503004(CBD-SR)
  • Lane8: K3503004(CBD-SR) with 1mM IPTG induction

Fire retardant test of K3503004

The test subject was in the size of bed sheet in 300*130mm and the average Moisture content% is 13.55%-14.37%. As demonstrated on Fig 3, all of our engineered flame retardant displayed an improvement compared with the water control.

Figure 3. Vertical Burning Test using bedsheet.
Figure 4. Flame retardancy test using wood.

As demonstrated in Fig 4, all of our engineered K3503001, K3503007 and K3507008 flame retardants displayed an improvement compared with the water control.