diff --git a/wiki/pages/notebook.html b/wiki/pages/notebook.html
index adbb55bf69f04487483b36133f62f83201ebef1f..40d97cd8a07c44e80f131a7f140c7965992e711d 100644
--- a/wiki/pages/notebook.html
+++ b/wiki/pages/notebook.html
@@ -109,8 +109,8 @@
We planned to add 0.1% arabinose to our LB medium for GFP carried by pGLO need arabinose to express GFP.
As the following figure:<br></p>
<div align="center">
- <img src="https://static.igem.wiki/teams/4702/wiki/description/notebook1.png"
- alt="pGLO plasmid design" style="width: 80%;">
+ <img src="https://static.igem.wiki/teams/4702/wiki/description/notebook1.png" alt="pGLO plasmid design"
+ style="width: 80%;">
</div><br>
<p>Sited from BIO-RAD:
<a href="https://www.bio-rad.com/zh-cn/applications-technologies/pglo-plasmid-map-resources?ID=NISQOC15">
@@ -649,7 +649,8 @@
<p>Additionally, 50 mL of the supernatant of 5 mM FeCl<sub>3</sub> induced culture after centrifugation
by 4000 rcf was evenly distributed into two tubes and centrifuged by 12000 rcf for 20 minutes.
Then the supernatant was discarded and resuspended with 5 mL protein purification buffer and 5 mL 7.5 M
- guanidine hydrochloride. After that it was kept on ice for 15 minutes. Samples are ultrafiltrated by using 100 kDa
+ guanidine hydrochloride. After that it was kept on ice for 15 minutes. Samples are ultrafiltrated by using
+ 100 kDa
ultrafiltration tubes and collected the flow through for TEM analysis.
<br>In all, six samples were prepared for TEM analysis, detailed treatment methods are shown below:
<br>
@@ -749,7 +750,7 @@
<p><b style="font-size: large;">TEM Sample Preparation</b></p>
<p>Participant(s): Shuoyi HU </p>
<p>
- Samples are resuspended by buffer A with 5 mM, 10mM, 15mM FeCl<sub>3</sub>
+ Samples are resuspended by buffer A with 5 mM, 10mM, 15mM FeCl<sub>3</sub>
induced cell lysate were prepared as samples for TEM analysis in 7/26.
</p>
<p></p>
@@ -889,16 +890,17 @@
<a href="https://www.atcc.org/products/htb-30#detailed-product-information">SK-BR-3 [SKBR3] - HTB-30
|
ATCC</a><br>
+ </p>
<p>Cell conditions are as below:</p>
- <div align="center"></div>
+ <div align="center">
<img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/sk-br-3-day1-7-29.jpg"
alt="sk-br-3-day1-7-29" width="300" height="">
- </p>
</div>
- <p align="right">Recorded by Yixiao XIAO</p>
</div>
+ <p align="right">Recorded by Yixiao XIAO</p>
</div>
</div>
+ </div>
<br>
<div class="card mx-auto">
<div class="card-header ani-card-header">
@@ -1049,11 +1051,15 @@
were transformed into SHuffle strain <i>E. coli</i> separately.
<br>(Named as SHuffle-antibody-cys-his and SHuffle-antibody-his-cys below)
</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-11-1.png"
- alt="Clones observed after pGLO-antibody-cys-his transformation" width="500" height=" ">
- <p></p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-11-2.png"
- alt="Clones observed after pGLO-antibody-his-cys transformation" width="500" height=" ">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-11-1.png"
+ alt="Clones observed after pGLO-antibody-cys-his transformation" width="500" height=" ">
+ <p></p>
+ </div>
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-11-2.png"
+ alt="Clones observed after pGLO-antibody-his-cys transformation" width="500" height=" ">
+ </div>
<p align="right">Recorded by Shuoyi HU</p>
</div>
</div>
@@ -1168,15 +1174,18 @@
tube, protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD from
top to bottom.
</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-19-1.png"
- alt="Antibody-cys-his protein purification result" width="600" height=" ">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-19-1.png"
+ alt="Antibody-cys-his protein purification result" width="600" height=" ">
+ </div>
<p>Sampling sequence of figure n+1: Tube No. 1-5 SHuffle-antibody-his-cys protein,
Tube No.5-8 SHuffle-antibody (Extracted from 8/15, only used as a supplementary
information), protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD,
17kD from top to bottom.</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-19-2.png"
- alt="Antibody-his-cys and antibody protein purification result" width="600" height=" ">
-
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-19-2.png"
+ alt="Antibody-his-cys and antibody protein purification result" width="600" height=" ">
+ </div>
<p align="right">Recorded by Shuoyi HU</p>
</div>
</div>
@@ -1222,9 +1231,10 @@
<p>Additionally, the vector containing scFV-streptavidin-his was transformed into SHuffle
strain <i>E. coli</i> separately. (Named as SHuffle-antibody-str-his below)
</p>
-
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-21-1.png"
- alt="Clones observed after SHuffle-antibody-str-his transformation" width="300" height=" ">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-21-1.png"
+ alt="Clones observed after SHuffle-antibody-str-his transformation" width="300" height=" ">
+ </div>
<p align="right">Recorded by Shuoyi HU </p>
</div>
<div class="vertical-line"></div>
@@ -1273,13 +1283,17 @@
<p>Sampling sequence of figure below: No.1-11 SHuffle-antibody-cys-his protein collection tube,
protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD from top to bottom.
</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-22-1.png"
- alt="Antibody-cys-his protein purification result" width="600" height=" ">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-22-1.png"
+ alt="Antibody-cys-his protein purification result" width="600" height=" ">
+ </div>
<p>Sampling sequence of figure below: Tube No. 2-12 SHuffle-antibody protein,
protein ladder: 180kD, 130kD, 95kD, 72kD, 55kD, 45kD, 34kD, 26kD, 17kD
from top to bottom.</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-22-2.png"
- alt="Antibody protein purification result" width="600" height=" ">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-22-2.png"
+ alt="Antibody protein purification result" width="600" height=" ">
+ </div>
<p>Additionally, as we found that under 5 mM FeCl<sub>3</sub> induction for biological
nanoparticle synthesis, there would have precipitated underlying in the culture
after centrifugation. To examine the nanoparticle we observed under TEM microscopy,
@@ -1395,11 +1409,15 @@
when a higher intensity of electron beam exposure applied). <br>And clear existence
of nanoparticle could be observed in the sample extracted from 5 mM
FeCl<sub>3</sub> induced culture.</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-25-1.png"
- alt="TEM result for negative control" width="600" height=" ">
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-25-2.png"
- alt="TEM result for for sample extracted from 5 mM FeCl<sub>3</sub> induced culture" width="600"
- height=" ">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-25-1.png"
+ alt="TEM result for negative control" width="600" height=" ">
+ </div>
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-25-2.png"
+ alt="TEM result for for sample extracted from 5 mM FeCl<sub>3</sub> induced culture" width="600"
+ height=" ">
+ </div>
<p>The expanded culture of SHuffle-antibody-str-his was collected through centrifugation
by 4000 rcf for 20 minutes and stored in -80 ºC for protein extraction in the evening.</p>
<p align="right">Recorded by Shuoyi HU</p>
@@ -1627,8 +1645,10 @@
4. Super, 5. pellet after dialysis, 6. sample4, 7. pellet, 8. Supernatant
after dialysis tube No. 3, 9. Supernatant after dialysis tube No. 4
</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-29-1.png"
- alt="Protein electrophoresis result" width="400" height=" ">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-29-1.png"
+ alt="Protein electrophoresis result" width="400" height=" ">
+ </div>
<p align="right">Recorded by Yanbo MAO </p>
</div>
<div class="vertical-line"></div>
@@ -1641,8 +1661,10 @@
<p>
Cell condition under 10x microscope as below:
</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-day1-10x-8-29.jpg"
- alt="bt474-10x-day1-8-29" width="280" height="">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-day1-10x-8-29.jpg"
+ alt="bt474-10x-day1-8-29" width="280" height="">
+ </div>
<p align="right">Recorded by Yixiao XIAO</p>
</div>
</div>
@@ -1670,9 +1692,11 @@
BCA test was conducted following the protocol of <a
href="{{ url_for('pages', page='experiments') }}#6">BCA test</a>.
Standard Curve of BCA test was shown below:</p>
- <img
- src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-30-bsa-standard-curve.png"
- alt="Standard Curve of BCA test" width="500" height=" ">
+ <div align="center">
+ <img
+ src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-8-30-bsa-standard-curve.png"
+ alt="Standard Curve of BCA test" width="500" height=" ">
+ </div>
<p></p>
<p>Using the standard curve, the concentration of the sample was calculated as below:</p>
<div class="shadow-lg p-3 mb-5 bg-white rounded">
@@ -1730,8 +1754,10 @@
to support the normal growth rate of BT-474 cells.</p>
<br>
<p>Cell condition under 10x microscope as below:</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt474-10x-day2-8-30.jpg"
- alt="bt474-10x-day2-8-30" width="280" height="">
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt474-10x-day2-8-30.jpg"
+ alt="bt474-10x-day2-8-30" width="280" height="">
+ </div>
<p align="right">Cell Experiment Recorded by Yixiao XIAO</p>
</div>
</div>
@@ -1817,8 +1843,11 @@
Cell Condition Record</b></p>
<p>Participant(s): Yixiao XIAO </p>
<p>Cell condition under 10x microscope as below:</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-10x-g2-day1-9-2.jpg"
- alt="bt-474-10x-g2-day1-9-2" width="300">
+ <div align="center">
+ <img
+ src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/cell/bt-474-10x-g2-day1-9-2.jpg"
+ alt="bt-474-10x-g2-day1-9-2" width="300">
+ </div>
<p align="right">Recorded by Yixiao XIAO</p>
</div>
</div>
@@ -2227,9 +2256,9 @@
<img
src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/notebook-9-9-antibody-final-used-on-website-tif.jpg"
alt="Antibody protein purification result" width="480" height=" " style="width: 70%;">
- </div>
+ </div>
</p>
-
+
<br> <br>
<p>Sampling sequence of the figure below: No.A03-A09 SHuffle-antibody-cys-his protein collection
tube, supernatant after centrifugation of broken cell culture, uninduced SHuffle-Antibody-cys-his,
@@ -2368,11 +2397,11 @@
href="{{ url_for('pages', page='experiments') }}#6">BCA
test</a>.
Standard Curve of BCA test was shown below:
- <div align="center">
- <img
- src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/result-for-notebook/10092023.png"
- alt="BCA standard curve in 9/10" width="480" height=" " style="width: 50%;">
- </div>
+ <div align="center">
+ <img
+ src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/result-for-notebook/10092023.png"
+ alt="BCA standard curve in 9/10" width="480" height=" " style="width: 50%;">
+ </div>
<p>Using the standard curve, the concentration of the sample was calculated as below:</p>
<div class="shadow-lg p-3 mb-5 bg-white rounded">
<div class="container w-80 h-100 mx-auto mt-3 mb-3">
@@ -2427,7 +2456,8 @@
<p align="right">Recorded by Shuoyi HU</p>
<p><b style="font-size: large;">Culture medium change</b></p>
<p>Participant(s): Yixiao XIAO </p>
- <p>BT-474 grew slowly but no bacteria infection had happened. Culture medium was changed with another 10ml medium with
+ <p>BT-474 grew slowly but no bacteria infection had happened. Culture medium was changed with another 10ml
+ medium with
20% of fbs to support the normal growth rate of BT-474 cells.
6ml of medium change was conducted for MDA-MB-231 cells.
</p>
@@ -2532,12 +2562,14 @@
<p>The spent medium was removed and both plates were washed of MDA-MB-231 and BT-474 with 3 ml PBS. Then,the
PBS solution was removed
and digested with 1 ml of trypsin/EDTA for about 10 min for BT-474 and 4 min for MDA-MB-231cells at
- 37°C. Then,
- the digestion was stopped with 3 ml of the corresponding culture medium for each kind of cells. And cells were centrifugated
+ 37°C. Then,
+ the digestion was stopped with 3 ml of the corresponding culture medium for each kind of cells. And cells
+ were centrifugated
at 1200
rpm for 3 min for BT-474 cells and 1000 rpm for MDA-MB-231 cells. The liquid supernatant was removed and
the
- cell pellet was resuspended with 6ml corresponding medium. Then, 10 ul of the cells suspension was taken out and mixed
+ cell pellet was resuspended with 6ml corresponding medium. Then, 10 ul of the cells suspension was taken
+ out and mixed
with 10ul
of trypan blue and counted the cell density and living rate, which were 7.17*10^5 cells/ml and 90.7% for
BT-474
@@ -2637,7 +2669,8 @@
</b>
</p>
<p>Participant(s): Yixiao XIAO, Songyuan XUE </p>
- <p>BT-474 grew slowly but no bacteria was infection had happened. Culture medium was changed with another 10ml medium with
+ <p>BT-474 grew slowly but no bacteria was infection had happened. Culture medium was changed with another
+ 10ml medium with
20% of fbs to support the normal growth rate of BT-474 cells.
6ml of medium change was conducted for MDA-MB-231 cells.
</p>
@@ -2670,7 +2703,8 @@
Participant: Yixiao XIAO
</p>
<p>After about 6 hours, all cells have adhered to the plate/dish wall,
- the spent medium was removed in each well and 100ul of prepared medium was added mixed with different reagents.
+ the spent medium was removed in each well and 100ul of prepared medium was added mixed with different
+ reagents.
</p>
<p>
Before drug treatment, cell conditions were recorded as below:
@@ -3006,9 +3040,11 @@
supernatant after centrifugation of broken cell culture of 18°C, collection tube No. A04, A05,
A06 of 18°C Culture.
</p>
- <img
- src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/marker-uninduced-18-c-sup-a04-a05-a06-b05-marker-37-c-uninduced-a05-a06-a07.jpg"
- alt="Antibody-cys-his protein purification result" width="480" height=" ">
+ <div align="center">
+ <img
+ src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/marker-uninduced-18-c-sup-a04-a05-a06-b05-marker-37-c-uninduced-a05-a06-a07.jpg"
+ alt="Antibody-cys-his protein purification result" width="480" height=" ">
+ </div>
<p align="right">Recorded by Shuoyi HU</p>
</div>
</div>
@@ -3039,9 +3075,12 @@
<a href="{{ url_for('pages', page='experiments') }}#5">BCA Protein Concentration Test</a>. <br>
Standard Curve of BCA test was shown below:
</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/0918-bca.png"
- alt="Antibody-cys-his protein concentration result" width="480" height=" ">
- <p>Using the standard curve, the concentrations of the samples were calculated as below: (the dillution factor
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/0918-bca.png"
+ alt="Antibody-cys-his protein concentration result" width="480" height=" ">
+ </div>
+ <p>Using the standard curve, the concentrations of the samples were calculated as below: (the dillution
+ factor
before BCA test was considered already) </p>
<div class="shadow-lg p-3 mb-5 bg-white rounded">
<div class="container w-80 h-40 mx-auto mt-3 mb-3">
@@ -3143,7 +3182,7 @@
<p>Participant(s): Shuoyi HU, Gehua WENG </p>
<p>The supernatant of 5 mM Fe<sub>2</sub>(SO<sub>4</sub>)<sub>3</sub> induced culture cell lysis were
filtrated through the 0.22 μm filtration membrane
- and then filtrated through 100 kD ultrafiltration membrane in 9/18, further
+ and then filtrated through 100 kD ultrafiltration membrane in 9/18, further
it was filtrated through 3kDa ultrafiltration membrane and collected the unfiltrated part.
<br><br>Later, after ultrasonication treatment, we conducted DLS analysis for the unfiltrated part.
The hydrodynamic size of the nanoparticle was 6.76 nm with an average baseline index 8.3, and the detailed
@@ -3300,13 +3339,17 @@
Subculture of MDA-MB-231 in 10 mm dish
</b></p>
<p>Participant(s): Yixiao XIAO </p>
- <p>The confluency of the MDA-MB-231 reached 90% and the subculture was conducted. The spent medium was removed
+ <p>The confluency of the MDA-MB-231 reached 90% and the subculture was conducted. The spent medium was
+ removed
and
- both plates of MDA-MB-231 were washed with 3 ml PBS. Then, the PBS solutionwas removed and digested with 1 ml of
- trypsin/EDTA for about 3 min at 37°C. Then, the digestion was stopped with 2 ml of the corresponding culture
+ both plates of MDA-MB-231 were washed with 3 ml PBS. Then, the PBS solutionwas removed and digested with 1
+ ml of
+ trypsin/EDTA for about 3 min at 37°C. Then, the digestion was stopped with 2 ml of the corresponding
+ culture
medium.
- And cells were centrifugated at 1200 rpm for 3 min. The liquid supernatant was removed and the cell pellets were resuspended
-
+ And cells were centrifugated at 1200 rpm for 3 min. The liquid supernatant was removed and the cell
+ pellets were resuspended
+
with 4 ml corresponding medium. For subculture, the seeding rate was 1:10 for MDA-MB-231 and 500 μl of
cell
suspension was added into two 100mm cell culture plate with 10 ml culture medium in each.</p>
@@ -3338,13 +3381,17 @@
aggregation, which could explained why the DLS analysis before gave much larger hydrodynamic diameter
than the diameter observed in TEM analysis.
<br><br> Below are two representative figures of the two samples:<br>
+ <div align="center">
<img
src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/0003-20230923-0936-57000-x-ceta-1.jpg"
alt="Chemically synthesized nanoparticle TEM result" width="480" height=" ">
- <p>Figure 1. Chemically synthesized nanoparticles' TEM result</p>
- <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/0023-20230923-1051-390-kx-ceta.jpg"
- alt="Biologically synthesized nanoparticle TEM result" width="480" height=" ">
- <p>Figure 2. Biologically synthesized nanoparticles' TEM result</p>
+ <p>Figure 1. Chemically synthesized nanoparticles' TEM result</p>
+ </div>
+ <div align="center">
+ <img src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/0023-20230923-1051-390-kx-ceta.jpg"
+ alt="Biologically synthesized nanoparticle TEM result" width="480" height=" ">
+ <p>Figure 2. Biologically synthesized nanoparticles' TEM result</p>
+ </div>
</p>
<p align="right">Recorded by Shuoyi HU</p>
<p><b style="font-size: large;">
@@ -3362,12 +3409,13 @@
<p>EDC/NHS Conjugation was conducted following the protocol of
<a href="{{ url_for('pages', page='experiments') }}#6">EDC/NHS Conjugation</a>.
- <br> 1)Double 0.036mg 8 µL of our chemical-synthesized nanoparticles and double 235µL newly bio-synthesized
+ <br> 1)Double 0.036mg 8 µL of our chemical-synthesized nanoparticles and double 235µL newly
+ bio-synthesized
nanoparticles(in Fe2(SO4)3) were transferred to four eppendorf tubes(EN PCn-ab5 1&EN PCn-ab5 2&EN
BNP-ab5
1&EN BNP-ab5 2) .
<br> 2)Then 100 µL 0.1M PBS was added to each of the tube to mix it thoroughly.
- <br> 3)Then, 20 µL of 0.2M EDC·HCl and 20 µL of 0.2M NHS were added to each tube.
+ <br> 3)Then, 20 µL of 0.2M EDC·HCl and 20 µL of 0.2M NHS were added to each tube.
<br> 4)Then 135 µL 0.1M PBS was added to first two tubes to make compensate for the solution volume.
<br> 5)After 10 min, 0.038mg antibody(AB-5) was added to each tube.
<br> 6)Then the tubes were stored in 4℃ .
@@ -3551,7 +3599,7 @@
Time Gradient
<br> Subculture of MDA-MB-231 and Sk-Br-3 in 10 mm
plates</b>
- <br>
+ <br>
</div>
<div class="card-body ani-card-body">
<p><b style="font-size: large;">
@@ -3569,24 +3617,31 @@
<br><br>
Representative figure of 48 hours resuspended cell pellet group was shown below:
<br>
+ </p>
+ <div align="center">
<img
src="https://static.igem.wiki/teams/4702/wiki/result-for-notebook/result-for-notebook/result-for-notebook/0005-20230925-2150-190-kx-ceta.jpg"
alt="TEM result of 48 hours resuspended cell pellet group" width="480" height=" ">
- <p>Figure 1. TEM result of 48 hours resuspended cell pellet group</p>
- </p>
+ <p>Figure 1. TEM result of 48 hours resuspended cell pellet group</p>
+ </div>
+
<p align="right">Recorded by Shuoyi HU</p>
<p><b style="font-size: large;">
Subculture of MDA-MB-231 and Sk-Br-3 in 10 mm plates</b></p>
<p>Participant(s): Yixiao XIAO </p>
<p>The confluency of the MDA-MB-231 reached 90% and Sk-Br-3 reached 75%-80% so that the subculture was
conducted.
- <br>The spent medium was removed and both plates of MDA-MB-231 and Sk-Br-3 were washed with 3 ml PBS. Then,
+ <br>The spent medium was removed and both plates of MDA-MB-231 and Sk-Br-3 were washed with 3 ml PBS.
+ Then,
the
PBS
- solution was removed and digested with 1 ml of trypsin/EDTA for about 3 min for MDA-MB-231 and 9 min for Sk-Br-3 at
- 37°C. Then, the digestion was stopped with 2 ml of the corresponding culture mediums. And cells were centrifuged at
+ solution was removed and digested with 1 ml of trypsin/EDTA for about 3 min for MDA-MB-231 and 9 min for
+ Sk-Br-3 at
+ 37°C. Then, the digestion was stopped with 2 ml of the corresponding culture mediums. And cells were
+ centrifuged at
1200
- rpm for 3 min. The liquid supernatant was removed and the cell pellet was resuspended with 4 ml corresponding
+ rpm for 3 min. The liquid supernatant was removed and the cell pellet was resuspended with 4 ml
+ corresponding
medium.
For subculture, the seeding rates were 1:10 for MDA-MB-231 and 1:4 for Sk-Br-3. Therefore, 500 μl of
MDA-MB-231 suspension and 1 ml of Sk-Br-3 suspension were added into two 100mm cell culture plates for
@@ -3595,22 +3650,22 @@
</p>
<p align="right">Recorded by Yixiao XIAO</p>
<p><b style="font-size: large;">
- EDC/NHS Linking</b></p>
- <p>Participant(s): Shijie HE</p>
+ EDC/NHS Linking</b></p>
+ <p>Participant(s): Shijie HE</p>
<br>1) 0.036mg 8 µL of our chemo-synthetic nanoparticles was transferred to an eppendorf tube(EN PCn) .
<br>2) Then 100 µL 0.1M PBS was added to each of the tube to mix it thoroughly.
<br>3) Then, 20 µL of 0.2M EDC·HCl and 20 µL of 0.2M NHS were added to each tube.
<br>4) Then 135 µL 0.1M PBS was added to first two tubes to make compensate for the solution volume.
<br>5) Then the tubes were stored in 4℃ .
- </p>
- <p align="right">Recorded by Shijie HE</p>
+ </p>
+ <p align="right">Recorded by Shijie HE</p>
</div>
</div>
<br>
<div class="card mx-auto">
<div class="card-header ani-card-header"> <b>9/26 Cell medium change
- <br> EDC-NHS Linking
- </b>
+ <br> EDC-NHS Linking
+ </b>
</div>
<div class="card-body ani-card-body">
<p>Participant(s): Yixiao XIAO</p>
@@ -3618,13 +3673,17 @@
plate).
<p align="right">Recorded by Yixiao XIAO</p>
<p><b style="font-size: large;">
- EDC/NHS Linking</b></p>
- <p>Participant(s): Shijie HE</p>
- <br>1) After 24 h, the solution (EN PCn) was transferred to 10kD ultrafiltration tubes and centrifuged for 10 min at 14000 g to remove the unbounded antibody.
- <br>2) Then 200 µL 0.1M PBS was added to the tubes to run 14000 g ultrafiltration for 10 min and wash the sample in this way twice.
- <br>3) Finally, the obtained approximate four 70 µL samples were collected in ultrafiltration tubes by reversing the inner tubes of ultrafiltration in new ultrafiltration tubes and running them at 1000g for 2 mins.
- </p>
- <p align="right">Recorded by Shijie HE</p>
+ EDC/NHS Linking</b></p>
+ <p>Participant(s): Shijie HE</p>
+ <br>1) After 24 h, the solution (EN PCn) was transferred to 10kD ultrafiltration tubes and centrifuged for
+ 10 min at 14000 g to remove the unbounded antibody.
+ <br>2) Then 200 µL 0.1M PBS was added to the tubes to run 14000 g ultrafiltration for 10 min and wash the
+ sample in this way twice.
+ <br>3) Finally, the obtained approximate four 70 µL samples were collected in ultrafiltration tubes by
+ reversing the inner tubes of ultrafiltration in new ultrafiltration tubes and running them at 1000g for 2
+ mins.
+ </p>
+ <p align="right">Recorded by Shijie HE</p>
</div>
</div>
<br>
@@ -3732,7 +3791,7 @@
number ratio of nanoparticle and antibody to be 1:2. For the
blank group, the same volume of buffer C was added. After reacting
for 1.5h under room temperature, proper amount of Buffer C was
- added and 14000g ultrafiltration in 10kDa tube was applied in
+ added and 14000g ultrafiltration in 10kDa tube was applied in
4℃ for at least 20 minutes to wash out unlinked antibodies.
After it was accomplished, the ultrafiltration tube was converted to
collect the left liquid by centrifuging at 1000g for 1 minute.
@@ -3812,14 +3871,18 @@
<p><b style="font-size: large;">
Preparation of Drug for Cell Treatment</b></p>
<p>Participant(s): Gehua WENG </p>
- <p>The spent medium was removed and both plates of MDA-MB-231 and Sk-Br-3 were washed with 3 ml PBS. Then, the
+ <p>The spent medium was removed and both plates of MDA-MB-231 and Sk-Br-3 were washed with 3 ml PBS. Then,
+ the
PBS
solution was removed and digested with 1 ml of trypsin/EDTA for about 10 min for Sk-Br-3 and 4 min for
MDA-MB-231cells
- at 37°C. Then, the digestion was stopped with 3 ml of the corresponding culture medium for each kind of cells. And
- cells were centrifuged at 1200 rpm for 3 min for Sk-Br-3 and MDA-MB-231 cells. The liquid supernatant was removed
+ at 37°C. Then, the digestion was stopped with 3 ml of the corresponding culture medium for each kind of
+ cells. And
+ cells were centrifuged at 1200 rpm for 3 min for Sk-Br-3 and MDA-MB-231 cells. The liquid supernatant was
+ removed
and
- the cell pellets were resuspended with 3 ml corresponding medium. Then, 10 ul of the cells suspension was taken
+ the cell pellets were resuspended with 3 ml corresponding medium. Then, 10 ul of the cells suspension was
+ taken
out
and mixed with 10ul of trypan blue and counted the cell density and living rate, which were
2.87*10<sup>6</sup>
@@ -3957,10 +4020,13 @@
Coomassie Brilliant Blue staining and the result is shown below, where the sampling
sequence is marker, MDA-MB-231 cell lysate, SK-BR-3 cell lysate, negative control
and scFv-cys-polyhis protein, the same as the sampling sequence of the other gel used for Western Blot.:
- <img
- src="https://static.igem.wiki/teams/4702/wiki/results/results/marker-mda-mb231-cell-lysate-sk-br-3-cell-lysate-neg-c-h-protein-tif.jpg"
- alt="Coomassie Brilliant Blue staining result" width="480" height=" ">
- <p>Figure 1. Coomassie Brilliant Blue staining result</p>
+
+ <div align="center">
+ <img
+ src="https://static.igem.wiki/teams/4702/wiki/results/results/marker-mda-mb231-cell-lysate-sk-br-3-cell-lysate-neg-c-h-protein-tif.jpg"
+ alt="Coomassie Brilliant Blue staining result" width="480" height=" ">
+ <p>Figure 1. Coomassie Brilliant Blue staining result</p>
+ </div>
The result showed that the relative concentration of scFv-cys-polyhis protein used for
binding induction is high.
<p align="right">Recorded by Shuoyi HU</p>
@@ -3988,7 +4054,8 @@
DLS analysis of the 8 samples prepared in 9/30 were conducted. We found that the
samples from the supernatant of cell lysate have low concentration of nanoparticles, and not
suitable for DLS analysis. While the samples from the resuspended cell lysate show high concentration.
- <br><br>In summary, we found that the optimized concentration of ferric ion for nanoparticle synthesis is 2.5
+ <br><br>In summary, we found that the optimized concentration of ferric ion for nanoparticle synthesis is
+ 2.5
mM,
as no significant difference in average hydrodynamic diameter and polydispersity index was found between
2.5
@@ -3998,15 +4065,19 @@
following
figures:
<br>
+ <div align="center">
<img src="https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/hydrodynamic-diameter-1.jpg"
alt="hydrodynamic summary figure" width="480" height=" ">
- <p>Figure 1. Hydrodynamic diameter summary figure</p>
+ <p>Figure 1. Hydrodynamic diameter summary figure</p></div>
+
+ <div align="center">
<img src="https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/polydispersity-1.jpg"
alt="polydispersity index summary figure" width="480" height=" ">
- <p>Figure 2. Polydispersity index summary figure</p>
+ <p>Figure 2. Polydispersity index summary figure</p></div>
+ <div align="center">
<img src="https://static.igem.wiki/teams/4702/wiki/results/dls-zeta-potential/zeta-potential-1.jpg"
alt="zeta potential summary figure" width="480" height=" ">
- <p>Figure 3. Zeta potential summary figure</p>
+ <p>Figure 3. Zeta potential summary figure</p></div>
<br>Raw datas of each samples could be found in the following pdf files.
<div class="card mx-auto text-center">
<div class="card-header ani-card-header">DLS Result of 0 mM FeCl<sub>3</sub> induced Sample</div>
@@ -4078,11 +4149,12 @@
<br>After washing the membrane for 30 minutes with TBS-T, finally the membrane
could be observed by adding exposing agent under the exposure machine.
<br><br>The protein bands were observed as shown below:<br>
+ <div align="center">
<img
src="https://static.igem.wiki/teams/4702/wiki/results/results/1002-scfv-c-h-0-3s-2023-10-02-11-54-53-ch-marker.jpg"
alt="Western Blot result" width="480" height=" ">
-
- <p>Figure 1. Western Blot result for scFv domain staining</p>
+
+ <p>Figure 1. Western Blot result for scFv domain staining</p></div>
Here only the standard protein control band could be observed to have been stained,
while the other bands were not stained. This may be due to the low concentration of protein
scontent in cell protein samples or poor binding affinity of the