Difference between revisions of "Part:BBa K3503004"
Alisaleong (Talk | contribs) |
|||
| (2 intermediate revisions by one other user not shown) | |||
| Line 31: | Line 31: | ||
margin: 0 auto; | margin: 0 auto; | ||
clear: both; | clear: both; | ||
| + | } | ||
| + | * { | ||
| + | box-sizing: border-box; | ||
| + | } | ||
| + | .column1 { | ||
| + | float: left; | ||
| + | width: 50%; | ||
| + | padding: 5px; | ||
| + | } | ||
| + | |||
| + | /* Clearfix (clear floats) */ | ||
| + | .row::after { | ||
| + | content: ""; | ||
| + | clear: both; | ||
| + | display: table; | ||
} | } | ||
</style> | </style> | ||
| Line 36: | Line 51: | ||
<h2>Sequencing and sequence alignment of K3503004</h2> | <h2>Sequencing and sequence alignment of K3503004</h2> | ||
| − | <img src=""> | + | <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 | <div class="legend">Figure 1. Representative sequencing results of K3503004 | ||
| Line 49: | Line 64: | ||
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>. | 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>. | ||
| − | <img src=""> | + | <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 | <div class="legend">Figure 2. Western Blot results of inducible expression of K1608000, K3503006, K3503004 | ||
| − | <br> | + | <br> |
<ul> | <ul> | ||
<li>Lane1: Bio rad protein dual color ladder; | <li>Lane1: Bio rad protein dual color ladder; | ||
| Line 65: | Line 90: | ||
<h2>Fire retardant test of K3503004</h2> | <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 | + | <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=""> | + | <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. | <div class="legend">Figure 3. Vertical Burning Test using bedsheet. | ||
| − | <img src=""> | + | <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. | <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 | + | <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> | </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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Sequencing and sequence alignment 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.
(a) Coomassie blue staining
(b) Western Blot
- 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.
As demonstrated in Fig 4, all of our engineered K3503001, K3503007 and K3507008 flame retardants displayed an improvement compared with the water control.