Team:SYSU-China/Project/Result/element test

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<!--正 文 部 分 开 始-->
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<DIV class="chapter">
<DIV class="chapter">
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<span>UPDATE <INS>09/22/2013</INS></span>
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<span>Project/Results/Parts Testing</span>
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<h1>Elements Testing</h1>
 
-
<a name="button02"></a><h2>The Cruel Guard:Suicide Gene and Its Ancillary Facility</h2>
 
-
<h3>1.Comparison between Suicide Genes: who is the most tough killer?</h3>
 
-
<p>
+
<a name="button02"></a><h1>Killer:suicide gene,the cruel guard</h1>
-
We collected several Suicide Genes which functions in different pathways and patterns. In order to choose one
+
<h2>Comparison between Suicide Genes: who is the most tough killer?</h2>
-
that is most capable of inducing apoptosis in cancer cell, we first carried out a parallel experiment to compare
 
-
 
-
their performance. The candidates were cloned into plasmid pcDNA3.0 in the MCS after CMV promoter just as shown
 
-
 
-
in picture A, and then transfected into HEK293(PEI) and HepG2(Lipo2000). A GFP processed in the same way was used
 
-
 
-
as a negative control to both indicate the transfection efficiency and normalize the death effect bring about by
 
-
 
-
transfection process.Pictures were taken 48 hr after the transfection process.
 
-
</p>
 
<p>
<p>
-
To achieve a better resolution of the apoptosis phenomenon, we tried two techniques with the 48hr cells. The
+
We have selected several Suicide Gene candidates which functions in different pathways and patterns. In order to choose one that is most capable of
-
first one is DAPI staining (done with RIP1 and RIP3), which …... The second one is PI staining and flow
+
mediating death in cancer cell, we first carried out a parallel experiment to compare their performance. The candidates were cloned into plasmid
-
cytometry counting(FCM counting), a technique that can give us the exact number of cell of different state. These
+
pcDNA3.0 in the MCS after CMV promoter just as shown in picture A, and then transfected into HEK293(PEI) and HepG2(Lipo2000). A GFP processed in  
-
 
+
-
two techniques were complementary to each other in a certain degree, for…
+
-
</P>
+
-
 
+
-
<div class="figure">
+
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/a/a0/Suicidegene-rip1.png" />
+
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/e/e5/Suicidegene-rip3.tif" />
+
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/6/64/Suicidegene-vp3.png" />
+
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/b/bc/Suicidegene-rip1_DAPI.png" />
+
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/e/ef/Hepg2_%E6%B5%81%E5%BC%8F%E5%87%8B
+
-
 
+
-
%E4%BA%A1%E7%8E%87_%E7%BB%9F%E8%AE%A1.png" />
+
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure1. Performance of different Suicide Genes
+
-
 
+
-
candidate in mediating apoptosis.</strong>(A).plasmids construction. (B,C) Wells transfected with RIP1\RIP3 both
+
-
 
+
-
showed significant phenotype of apoptosis compared with GFP control, 48 hr after transfection. After DAPI
+
-
 
+
-
staining, dead cell showed deep blue color and the phenotype was better visualized. (D)Apoptosis phenotype of
+
-
 
+
-
Apoptin(VP3). The protein was expressed in fusion with a GFP. Compared with 0 hr , apoptosis rose 48 hr after
+
-
 
+
-
transfection, and GFP vision indicated the transfection efficiency.(E)DAPI staining of RIP1, zoomed in vision. We
+
-
 
+
-
can see that the dead cell is…(F).PI staining and FCM data.</p>
+
-
<div class="clear"></div></div>
+
-
 
+
-
<p>
+
-
The figure shows that Both RIP1 and RIP3 had a outstanding performance in mediating apoptosis. Although Apoptin
+
-
 
+
-
also killed cell in the results, its performance wasn’t stable since we cannot reproduce the results with
+
-
 
+
-
another construction( data not shown.) However, according to many paper, this is a powerful protein with a
+
-
 
+
-
specificity in killing cancer cell, which persuade us to keep working with it in the latter experiments.
+
-
 
+
-
Moreover, we also test Bax/Bax S184A with the same method. However, no significant phenotype could be observed
+
-
 
+
-
(data not shown) and thus we abandoned them in our Suicide Gene list.
+
-
</p>
+
-
<p>
+
-
The PI staining and FCM data showed that the apoptosis rate of RIP1 and RIP3 were about …. However, there could
+
-
be some problems about the results such as a low expression level of GFP control, which made the data not so
+
the same way was used as a negative control to both indicate the transfection efficiency and normalize the death effect bring about by transfection
-
convincing. And we had planned a repeat for the experiment.
+
process.Pictures were taken 48 hr after the transfection process.
</p>
</p>
-
<h3>2.Suicide Gene working with its ancillary facility</h3>
 
<p>
<p>
-
Although we have successfully proved that each device of our pathway worked well, we wanted to further make use
+
To achieve a better resolution of the lethal effect, we tried two techniques with the 48hr cells. The first one is DAPI staining done in RIP1 and
-
of the convenience of transient transfection due to the difficulty and time-consuming quality of Lenti-Virus
+
RIP3 groups. DAPI can specificly binds to DNA but is rejected by living cells to some extent. With DAPI staining, more cells were darkly dyed in
-
transfection. So we designed combinational experiments, centered on our protagonist, the Suicide Gene, to further
+
the group of RIP1 and RIP3 compared to GFP control. Broken nuclei membrane can be noticed in cells transfected with RIP1 because DAPI blue spreaded
-
confirmed the devices’ performance in working coordinately. Although we didn’t have enough time to finished
+
all over cytopkasm The second one is flow cytometrycounting(FCM counting). Cells were stained by PI and then analysed by FCM counting. Dead cells
-
every one of them before Regional Jamboree, we have put them into our schedule of October if they’re still
+
binded more PI per cell in a similar reason to DAPI staining. These two techniques were complementary to each other in a certain degree, for DAPI
-
needed at that time(in situation where we still don’t have enough convincing data from Lenti-Virus work.)
+
is superior in morphological observation while FCM counting is good at counting.
-
</p>
+
</P>
-
<p>
+
-
<h4>A.Controlling the switch: a combination of Suicide Gene and Tet-off system.</h4>
+
-
</p>
+
-
<p>
+
-
Tough as the Suicide Gene is, what we expect from it was to protect instead of killing the innocent. And the 2
+
-
 
+
-
kinds of “innocent” cells in our designing were iPSC and Heptocyte. For iPSC, we have assigned it with a
+
-
 
+
-
controlling panel: the Tet-Off system. Thus we designed an experiment to test the ability of Tet-off system to
+
-
 
+
-
drive or turning off the expression of Suicide Gene. The designing and expected results were both shown below and
+
-
 
+
-
Tet-Off construction driving GFP expressiong was taken as the positive control.
+
-
</p>
+
<div class="figure">
<div class="figure">
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
<img class="fig_img" width="750px" src="https://static.igem.org/mediawiki/2013/3/31/Figure-1.png" />
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure2.</strong>apoptosis pathway induced by
+
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 1. Performance of different Suicide Genes candidates in causing
-
hBax</p>
+
apoptosis.</strong>(A).plasmids construction.(B)Apoptosis phenotype of Apoptin(VP3). The protein was expressed in fusion with a GFP. Compared with
-
<div class="clear"></div></div>
+
-
<h4>B.Rescue scheme: a combination of Suicide Gene and MicroRNA-Target system.</h4>
+
0 hr , apoptosis rose 48 hr after transfection, and GFP vision indicated the transfection efficiency.(C,D) Wells transfected with RIP1\RIP3 both
-
<p>
+
-
For Heptocyte the protection comes from functioning of MicroRNA-Target system since the miR122 level was very
+
-
high in Heptocyte(see Design). Due to technical bottleneck in transfection of Heptocyte, we decided to first
+
showed significant phenotype of apoptosis compared with GFP control, 48 hr after transfection. After DAPI staining, dead cell showed deep blue
-
simulated the environment and thus designed a mini-orthogonal experiment crossing variables of Target and miR122.  
+
color and the phenotype was better visualized.(E)DAPI staining of RIP1, zoomed in vision.We can clearly observe boundary of cells which indicate a
-
The designing and expected results were shown below.
+
broken nucleus membrane and DNA spread all over the cells(F,G).PI staining and FCM data of our Suicide Gene. </p><div class="clear"></div></div>
-
</p>
+
-
<p>
+
-
The results, together with the RT-qPCR data of miR122 level in Heptocyte(see。。。。), would prove that the
+
-
device’s knockdown efficiency is sufficient to protect Heptocyte. Also with the mathematical model derived from
 
-
 
-
Simulation experiment with GFP(see MicroRNA-Target part and Modelling), we can predict the rescue efficiency
 
-
 
-
under different concentration of miR122 and target-site number.
 
-
</p>
 
-
 
-
<div class="figure">
 
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
 
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure3.</strong>apoptosis pathway induced by
 
-
 
-
hBax</p>
 
-
<div class="clear"></div></div>
 
-
 
-
<h4>C.The final test-run: combination of all three systems before setting up stable cell line</h4>
 
<p>
<p>
-
With a idealized transfection efficiency, theoretically the three systems can be assembled and tested by simply
+
The figure shows that Both RIP1 and RIP3 had a outstanding performance in mediating apoptosis. Although Apoptin also killed cell in the results,
-
transfect the cells with 3 plasmids(fig. ..). Plasmids A and B can turn on the Tet-off system and drive
+
its performance wasn’t stable since we cannot reproduce the results with another construction( data not shown.) However, according to many paper,
-
expression of GOI(Suicide Gene or GFP), while plasmid C can simulate the environment of Hepatocyte and execute
+
this is a powerful protein with a specificity in killing cancer cell, which persuade us to keep working with it in the latter experiments.
-
the rescue scheme. Although practically it could be difficult due to efficiency and duration time limitation, we  
+
Moreover, we also test Bax/Bax S184A with the same method. However, no significant phenotype could be observed(data not shown) and thus we  
-
still decided to try it as a complement and see how much we can do with the transient transfection strategy. So
+
abandoned them in our Suicide Gene list.
-
 
+
-
we designed the following orthogonal experiment which, though seemingly complicated, could perfectly give a
+
-
 
+
-
cross-reference to each variable and confirm all elements of our pathway.
+
</p>
</p>
-
 
-
<div class="figure">
 
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
 
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure4.</strong>apoptosis pathway induced by
 
-
 
-
hBax</p>
 
-
<div class="clear"></div></div>
 
<p>
<p>
-
We carried out the plan in HEK293 cell with PEI. However, due to technical problems no obvious results can be
+
The PI staining and FCM data showed that the apoptosis rate of RIP1 and RIP3 were about 51.7% and 47.6%, which were significantly higher than the
-
observed. We may try to optimize the technical factors to promote efficiency and repeat the experiment if
+
GFP control but rather lower than resent studies. However, this difference may be caused by the relatively low transfection efficiency in Heg G2.
-
necessary.
+
More details link to <a href="https://2013.igem.org/Team:SYSU-China/Project/Modeling">Modeling</a>. 
</p>
</p>
-
<a name="button01"></a><h2>Simulation Testing of MicroRNA-Target Devices: Security Gate for Liver Cell</h2>
+
<a name="button01"></a><h1> Sensor:miRNA binding site, the security gate for liver cell</h1>
-
<h3>1.Performance comparison between target-sites: CULT and Complete</h3>
+
<h2>Performance comparison between binding sites: CULT and Complete</h2>
<p>
<p>
-
Two kinds of target-sites were taken into consideration just as mentioned in module Design. To make a comparison  
+
Two kinds of binding site were taken into consideration just as mentioned in module Design. To make a comparison between their knockdown efficiency  
-
 
+
-
between their knockdown efficiency under different concentration of miR122, another orthogonal experiment was
+
-
performed and two plasmids(fig.A) were co-transfected by PEI into HEK293 cell line. Pictures were taken 24 hours
+
under different concentration of miR122, another orthogonal experiment was performed and two plasmids(fig2.A) were co-transfected by PEI into  
-
after transfection.
+
HEK293 cell line. Pictures were taken 24 hours after transfection.
</p>
</p>
<div class="figure">
<div class="figure">
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
<img class="fig_img" width="300px" src="https://static.igem.org/mediawiki/2013/e/ef/SYSU-Figure5-A-construction.png" />
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure5. Comparison of performance of different  
+
<img class="fig_img" width="400px"style="float:right;" src="https://static.igem.org/mediawiki/2013/0/05/SYSU-Figure5-B-comparison_ed.png" />
 +
<p class="des" style="margin-top:0px;width:300px"><strong>Figure 2. Comparison of performance of different binding site in knocking down GFP
-
target-sites in knocking down GFP expression. </strong>(A) Plasmids A/B/C/D produced mRNA of GFP with different  
+
expression. </strong>(A) Plasmids A/B/C/D produced mRNA of GFP with different binding site in its 3’ UTR. Plasmids E produced miR122. Plasmids
-
target-site in its 3’ UTR. Plasmids E produced miR122 by strategy of . Plasmids used in this experiment had the  
+
used in this experiment had the problem of producing a background noise of GFP, which was solved later(see below).(B) Knockdown effect of different
-
problem of producing a background noise of GFP, which was solved later(see below).(B) Knockdown effect of
+
binding site under different miR122 level. The fields of vision were chosen so that they could well represent the average level of the wells.</p>
-
 
+
-
different target site under different miR122 level. The fields of vision were chosen so that they could well  
+
-
 
+
-
represent the average level of the wells.</p>
+
<div class="clear"></div></div>
<div class="clear"></div></div>
<p>
<p>
-
The result shows that with the same copy number, complete target-site works better than CULT target-site, which  
+
The result shows that with the same copy number, complete binding site works better than CULT binding site, which fits the results of the <a href=" https://2010.igem.org/Team:Heidelberg/Parts">2010 Heiderburg iGEM team</a>. What’s more, two copies binding site works better than single site, which encourage us to further build binding site of more
-
fits the results of the 2010 Heiderburg iGEM team. What’s more, two copies target-site works better than single
+
copy number and employed more quantitating experiments.
 +
</p>
-
site, which encourage us to further build target-sites of more copy number and employed more quantitating
 
-
 
-
experiments.
 
-
</p>
 
<p>
<p>
-
Plasmid E used in the experiment produced background GFP, which was a problem neglected by us. After finding out  
+
Plasmid E used in the experiment produced background GFP, which was a problem neglected by us. After finding out the problem, we did quick-change
-
the problem, we did quick-change to introduce a stop-codon into the CDS of GFP, which proves very efficient. The  
+
to introduce a stop-codon into the CDS of GFP, which proves very efficient. The new miR122 expression plasmids were applied to other experiments
-
new miR122 expression plasmids were applied to other experiments just as mentioned. However, we found that  
+
just as mentioned. However, we found that although this did interfere our results to some degree, we could still gain insight into the problem so
-
although this did interfere our results to some degree, we could still gain insight into the problem so the data  
+
the data was kept.
-
 
+
-
was kept.
+
</p>
</p>
-
<h3>2.Quantitative characterization and target-site modelling</h3>
+
<h2>Quantitative characterization and binding site modelling</h2>
<p>
<p>
-
In order to build a precisely controlled device, we decided to further characterized the complete-target-sites of  
+
In order to build a precisely controlled device, we decided to further characterized the complete binding site of different copy number and chose  
-
 
+
-
1\2\4 copy number and chose the most suitable one for pathway construction. To begin with we design another
+
-
orthogonal experiment crossing the two variables, target-site-number N and MiR122 concentration M, with a more
+
the most suitable one for pathway construction. To begin with we designed another orthogonal experiment crossing the two variables, binding site  
-
precise gradients for MiR122. But soon we found that it would be too labour-costing and decided that a better way
+
number N and MiR122 concentration M, with a more precise gradients for MiR122. But soon we found that it would be too labour-costing and decided  
-
was to replace it with 2 dosage-gradient experiments and a mathematic model. (fig.
+
that a better way was to replace it with 2 dosage-gradient experiments and a mathematic model. (fig.3)
</p>
</p>
<div class="figure">
<div class="figure">
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/d/d0/SYSU-Figure6.png" />
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure6.</strong>apoptosis pathway induced by
+
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 3.Two strategies of characterizing miR122 binding site system.</strong>We adapt
-
hBax</p>
+
the latter strategy,making use of modeling principle,to simplify the work.</p>
<div class="clear"></div></div>
<div class="clear"></div></div>
-
<h4>A.Dosage experiment testing performance of 2 copies complete target-site</h4>
+
<h3>A.Dosage experiment testing performance of 2 copies complete binding site</h3>
<p>
<p>
-
Firstly we fixed the target-site number at 2 and set up a concentration gradient for miR122 expression plasmid
+
Firstly we fixed the binding site number at 2 and set up a concentration gradient for miR122 expression plasmid (fig.7,A, plasmid CEP) to gain
-
(fig. A, plasmid B) to gain insight into its dosage effect. Plasmids A and B were co-transfected using PEI into  
+
insight into its dosage effect. Plasmids A and B were co-transfected using PEI into HEK293 cell line. The gradient were set up from 0 to 0.5ug of
-
HEK293 cell line. The gradient were set up from 0 to 0.5ug of plasmid per well. The experiment was done with 2  
+
plasmid per well. The experiment was done with 2 parallel groups of wells( that is, 6 wells as a group and there were 2 repeats) to leave samples
-
parallel groups of wells( that is, 6 wells as a group and there were 2 repeats) to leave samples for both  
+
for both Western-Blot and RT-qPCR(since both techniques require destruction of sample). To ensure the parallelity so that the two data can be
-
Western-Blot and RT-qPCR(since both techniques require destruction of sample). To ensure the parallelity so that
+
employed to illustrate the problem together, we had repeated the experiment 2 times till we gained same fluorescence intensity with corresponding
-
the two data can be employed to illustrate the problem together, we had repeated the experiment 2 times till we
+
wells(data not shown).
-
 
+
-
gained same fluorescence intensity with corresponding wells(data not shown).
+
</p>
</p>
<div class="figure">
<div class="figure">
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
<img class="fig_img" width="750px" src="https://static.igem.org/mediawiki/2013/d/d5/Figure-4.png" />
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure7.Dosage effect of miR122 on 2 copy number  
+
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 4. Dosage effect of miR122 on 2 copy number  
-
target-site.</strong>(A)Plasmid A produced GFP mRNA with a 2 copies Complete target-site in its 3’ UTR, while  
+
binding site.</strong>(A)Plasmid CG2T produced GFP mRNA with a 2 copies Complete binding site in its 3’ UTR,while  
-
plasmid B produced miR122 without making fluorescence noise. (B) A gradient of total plasmid amount per well was  
+
plasmid CEP produced miR122 without making fluorescence noise. (B)A gradient of total plasmid amount per well was  
-
set up as shown. A significant positive correlation between knockdown efficiency and plasmid B concentration can  
+
set up as shown. A significant positive correlation between fluorescence knockdown efficiency and plasmid CEP concentration can  
-
be observed.(C) RT-qPCR showed that plasmid B successfully express miR122 in cells, the level of which was in a  
+
be observed.(C) RT-qPCR showed that plasmid CEP successfully express miR122 in cells, the level of which was in a  
-
good linear correlation with the transfection concentration through linear-regression analysis(data not shown).
+
good linear correlation with the transfection concentration through linear-regression analysis(see Heptocyte).
-
(D).A not-so-successful Western-Blot showing the GFP level in each well. Based on scanning densitometry, the
+
(D).Western-Blot showing the GFP level in each well.(E).GFP expression level derived by scanning densitometry,which is in good correlation with
-
level can be counted(chart ) and plotted as above.</p>
+
fluorescence observation. </p>
<div class="clear"></div></div>
<div class="clear"></div></div>
-
 
-
knockdown efficiency chart <br />
 
-
<p>收样时间:48hr<br />Target-site: Complete 2-copies</p>
 
-
<table width="536">
 
-
<tr>
 
-
<td width="200">Mir122</td>
 
-
<td width="50">1</td>
 
-
<td width="50">4</td>
 
-
<td width="50">10</td>
 
-
<td width="50">28</td>
 
-
<td width="50">76</td>
 
-
<td width="50">161</td>
 
-
</tr>
 
-
<tr>
 
-
<td>GFP(Western Blot)</td>
 
-
<td></td>
 
-
<td></td>
 
-
<td></td>
 
-
<td></td>
 
-
<td></td>
 
-
<td></td>
 
-
</tr>
 
-
</table>
 
<p>
<p>
-
The results showed a simple and elegant linear correlation between knockdown efficiency and miR122 concentration.  
+
The results showed a simple and elegant linear correlation between knockdown efficiency and miR122 concentration.What's more, we have planned to  
-
 
+
-
Due to some technical problems with Western-Blot the data from well of 0.5ug plasmid B wasn’t consistent with
+
-
fluorescence observation, and we have planned to reconfirmed the data.
+
repeat the experiment and make a statistic data.
</p>
</p>
-
<h4>B.Performance of complete target-site of different copy number</h4>
+
<h3>B.Performance of complete binding site of different copy number</h3>
<p>
<p>
-
Secondly we fixed the miR122 concentration at 0.75ug to ensure an optimal performance of each targets and let  
+
Secondly we fixed the miR122 concentration at 0.75ug to ensure an optimal performance of each binding sites and let  
-
target-site number be the variable this time. Since we had already made sure of the relationship of miR122 level  
+
binding site number be the variable this time. Since we had already made sure of the relationship of miR122 level  
and plasmid concentration, RT-qPCR was no longer needed and so was the parallel groups.  
and plasmid concentration, RT-qPCR was no longer needed and so was the parallel groups.  
</p>
</p>
<p>
<p>
-
Apart from the 2 and 4 copy target-site, we have also constructed a mixed-up plasmid with 2 complete target-site  
+
Apart from the 2 and 4 copy binding site, we have also constructed a mixed-up plasmid with 2 complete binding site  
-
followed by 2 CULT target-site(2+2).
+
followed by 2 CULT binding site(2+2).
</p>
</p>
<div class="figure">
<div class="figure">
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
<img class="fig_img" width="750px" src="https://static.igem.org/mediawiki/2013/3/32/Figure-5.png" />
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure8.</strong>apoptosis pathway induced by
+
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 5. Comparison of knockdown efficiency of different binding site.</strong>
-
hBax</p>
+
(A).Plasmids construction for different binding site.(B).Knockdown effect of binding sites are significant in fluorescence vision.It can be
-
<div class="clear"></div></div>
+
-
Knock down efficiency chart<br />
+
observed that the 4*complete binding site has the most robust performance.(C).Western Blot of the experiment.The lanes has been cutted to omit some
-
<p>Mir122 concentration:0.75ug=</p>
+
unrelated data.(D).Knockdown efficiency of the binding sites counted by data derived from Western Blot.</p>
-
<table width="536">
+
<div class="clear"></div></div>
-
<tr>
+
-
<td width="200">Target Site Number</td>
+
-
<td width="75">0</td>
+
-
<td width="75">1</td>
+
-
<td width="75">2</td>
+
-
<td width="75">4</td>
+
-
</tr>
+
-
<tr class="first-table-line">
+
-
<td>GFP(Western Blot)</td>
+
-
<td></td>
+
-
<td></td>
+
-
<td></td>
+
-
<td></td>
+
-
</tr>
+
-
</table>
+
<p>
<p>
-
The results show that 4 copy target-site has the most brilliant knock-down efficiency, which is about under such  
+
The results show that 4 copy binding site has the most brilliant knock-down efficiency, which is about under such  
a miR122 level, which conforms to the data given by 2010 Heiderburg using dual-fluorescent system. What’s more,  
a miR122 level, which conforms to the data given by 2010 Heiderburg using dual-fluorescent system. What’s more,  
-
in Part1 we have already proved that 2 copy target-site works better than 1 copy target-site. Finally we decided  
+
in Part1 we have already proved that 2 copy binding site works better than 1 copy binding site. Finally we decided  
-
to first continue further construction of the pathway with the 2 copy target-site, considering that its moderate  
+
to first continue further construction of the pathway with the 2 copy binding site, considering that its moderate  
knockdown efficiency may well coordinate with the other two systems. And if the efficiency turns out improper, we  
knockdown efficiency may well coordinate with the other two systems. And if the efficiency turns out improper, we  
Line 405: Line 252:
</p>
</p>
-
<h4>C.Modelling</h4>
+
<h3>C.Modeling</h3>
<p>
<p>
-
In order to better understand the quality of our miR122-Target system, we have employed interpolation technique  
+
In order to better understand the quality of our miR122 binding site system, we have employed interpolation technique  
with Matlab to model its performance. The modeling can be seen at Modelling module in Results. Also, we have  
with Matlab to model its performance. The modeling can be seen at Modelling module in Results. Also, we have  
-
planned a more well-designed experiment to test and characterize all target-sites(in fact, there are 8 of them,  
+
planned a more well-designed experiment to test and characterize all binding site(in fact, there are 8 of them,  
produced by permutation and combination of different numbers of complete and CULT). The data derived from that  
produced by permutation and combination of different numbers of complete and CULT). The data derived from that  
Line 420: Line 267:
<div class="figure">
<div class="figure">
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/e/ee/SYSU-Figure9_ed.png" />
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure8.</strong>apoptosis pathway induced by
+
<p class="des" style="margin-top:0px;width:350px"><strong>Figure 6. Modeling of MiR122 binding site system.</strong></p>
-
 
+
-
hBax</p>
+
<div class="clear"></div></div>
<div class="clear"></div></div>
-
<a name="button03"></a><h2>Simulation testing of Tet-Off system: Controlling panel for iPSC</h2>
+
 
 +
<a name="button03"></a><h1>Switch:Tet-Off system,the controlling panel for iPSC</h1>
<p>
<p>
We also used eGFP to test the switching and driving performance of Tet-Off System, which would be a convenient  
We also used eGFP to test the switching and driving performance of Tet-Off System, which would be a convenient  
Line 436: Line 282:
<div class="figure">
<div class="figure">
-
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/2/2f/Which_suicide_gene_fig_1.gif" />
+
<img class="fig_img" height="200px" src="https://static.igem.org/mediawiki/2013/2/22/SYSU-Figure10-A.png" />
-
<p class="des" style="margin-top:200px;width:300px"><strong>Figure9. Performance of Tet-off system in switching  
+
<img class="fig_img" height="200px" src="https://static.igem.org/mediawiki/2013/f/f2/SYSU-Figure10-B-tet_off_ed.png" />
 +
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 7. Performance of Tet-off system in switching  
-
between different state.</strong>Two plasmids(fig.) were co-transfected into Bosc cell line using PEI. The wells  
+
between different state.</strong>Two plasmids(fig.7.A) were co-transfected into Bosc cell line using PEI. The wells  
with only plasmids B/C or D characterized the basal leaky expression of different pTRE elements, which can be  
with only plasmids B/C or D characterized the basal leaky expression of different pTRE elements, which can be  
Line 463: Line 310:
and leave the other ones as alternative schemes.
and leave the other ones as alternative schemes.
</p>
</p>
-
<a name="button04"></a><h2> Lenti-virus</h2>
+
<p>
 +
In addition, we have received another version of Tet-off system in which the regulatory protein is fused by TetR and KRAB. According to our test
 +
 
 +
result, the KRAB Tet-off system has the best fold increase but its leakage effect is higher the pTight. Maybe the leakage effect can be further
 +
 
 +
eliminated by carefully select the optimal clone in stable cell lines. However, we abandon this system because KRAB theoretically does not work
 +
 
 +
well in ES cells for our usage in iPSCs .
 +
</p>
 +
<p>
 +
<div class="figure">
 +
<img class="fig_img" height="200px" src="https://static.igem.org/mediawiki/2013/8/8f/SYSU-Figure11-KRAB.png" />
 +
<p class="des" style="margin-top:150px;width:350px"><strong>Figure 8. KRAB the new tet off system</strong></p>
 +
<div class="clear"></div></div>
 +
 
 +
</p>
 +
 
 +
<h2>  Further test for parts combination</h2>
 +
<p>Since we have successfully proved that each part of our pathway worked well, we even wanted to further prove our device making use of the
 +
 
 +
convenience of transient transfection, given that Lenti-Virus infection is too time-consuming. So we designed combinational experiments, centered
 +
 
 +
on our protagonist, the Suicide Gene, to further confirmed the parts’ performance in working coordinately, including the test for combination of
 +
 
 +
Suicide gene and tet-off system, the test for combination of Suicide Gene and miRNA binding site system and the test for the whole device. However,
 +
 
 +
because of the time limited, we have to spend more energy on learning and researching the technology of stable cell lines establishment and assay,
 +
 
 +
we finally did not choose to finish every experiments before Regional Jamboree.
 +
<div class="figure">
 +
<img class="fig_img" height="200px" src="https://static.igem.org/mediawiki/2013/7/77/SYSU-Figure2-A-construction.png" />
 +
<img class="fig_img" height="200px" src="https://static.igem.org/mediawiki/2013/8/8a/SYSU-Figure2-B_%282%29.png" />
 +
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 9. Combinational test: Tet-Off system and Suicide Gene</strong></p>
 +
<div class="clear"></div></div>
 +
 
 +
<div class="figure">
 +
<img class="fig_img" height="220px" src="https://static.igem.org/mediawiki/2013/4/4d/SYSU-Figure3-A-construction.png" />
 +
<img class="fig_img" height="220px" src="https://static.igem.org/mediawiki/2013/7/7d/SYSU-Figure3-B_%282%29.png" />
 +
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 10. Rescue experiment:miR122 binding site system and Suicide Gene</strong></p>
 +
<div class="clear"></div></div>
 +
 
 +
<div class="figure">
 +
<img class="fig_img" height="250px" src="https://static.igem.org/mediawiki/2013/1/1e/SYSU-Figure4-A-construction.png" />
 +
<img class="fig_img" height="300px" src="https://static.igem.org/mediawiki/2013/8/80/SYSU-Figure4-B_%282%29.png" />
 +
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 11. Orthogonal experiment of all three devices.</strong></p>
 +
<div class="clear"></div></div>
 +
 
 +
<a name="button04"></a><h1> Lentivirus</h1>
 +
<h2>Packaging: </h2>
 +
<p>We packaged two batches of lentivirus, one containing the upstream tTA plasmid, the other containing the downstream suicide gene and binding
 +
 
 +
site
 +
 
 +
plasmid. </p>
 +
<div class="figure">
 +
<img class="fig_img" width="700px" src="    https://static.igem.org/mediawiki/2013/5/51/SYSU-Result-lenti1.png
 +
      " />
 +
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 12. </strong> The map of plasmids for lentivirus packaging.        </p>
 +
<div class="clear"></div></div>
 +
 
 +
<p>The package of lentivirus vectors was done in HEK 293T cell lines in 10cm cell culture plates, along with two packaging plasmid: psPAX2 and
 +
 
 +
pMD2G.
 +
 
 +
Both of them were 3rd generation packaging plasmid. We then collected the CM supernatant after 48h and 72h, and transfered all of them into tubes.
 +
 
 +
</p>
 +
 
 +
<h2>Centrifugation: </h2>
 +
<p>Because these lentivirus vectors were supposed to infect not only Hep G2 cell lines but also mouse iPSCs, which was very difficult to infect,
 +
 
 +
the
 +
 
 +
requirement of the MOI (Multiplicity of Infection) was much higher than normal experiments, so before infecting cells, we ultracentrifugated these
 +
 
 +
viral vectors.</p>
 +
 
 +
<div class="figure">
 +
<img class="fig_img" width="700px" src="    https://static.igem.org/mediawiki/2013/3/3c/SYSU-Result-lentivirus.png " />
 +
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 13.</strong> The steps for lentivirus packaging and centrifugation.        </p>
 +
<div class="clear"></div></div>
 +
 
 +
<p>Here is the photo of the 48hr culture medium with lentivirus.</p>
 +
 
 +
<div class="figure">
 +
<img class="fig_img" width="700px" src="    https://static.igem.org/mediawiki/2013/4/4c/Virus.jpg" />
 +
<p class="des" style="margin-top:0px;width:700px"><strong>Figure 14.</strong> The colors of culture medium with different lentivirus.  </p>
 +
<div class="clear"></div></div>
 +
 
 +
 
 +
<p>After successfully testing for each part, it's the right time to think about the assays in different period of cells. </p>
 +
<p><strong>Click the cells to see the results.</strong></p>
 +
 
 +
<div class="results_guide2">
 +
<a class="guide_button2" id="ips" href="https://2013.igem.org/Team:SYSU-China/Project/result/stable_assay?#button01"></a>
 +
<a class="guide_button2" id="hepatocyte" href="https://2013.igem.org/Team:SYSU-China/Project/result/stable_assay?#button02"></a>
 +
<a class="guide_button2" id="hepatoma" href="https://2013.igem.org/Team:SYSU-China/Project/result/stable_assay?#button03"></a>
 +
</div>
 +
 
-
<DIV id="references">
 
-
<h3>References</h3>
 
-
<p><a class="references">[1]</a>Hui Xu etc. Liver-Enriched Transcription Factors Regulate MicroRNA-122 That
 
-
Targets CUTL1 During Liver Development.</p>
 
-
<p><a class="references">[2]</a>Tet-Off and Tet-On Gene Expression Systems User Manual</p>
 
-
</DIV>
 
<!--正 文 部 分 结 束-->
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Latest revision as of 03:41, 29 October 2013

ipsc

Project/Results/Parts Testing

Killer:suicide gene,the cruel guard

Comparison between Suicide Genes: who is the most tough killer?

We have selected several Suicide Gene candidates which functions in different pathways and patterns. In order to choose one that is most capable of mediating death in cancer cell, we first carried out a parallel experiment to compare their performance. The candidates were cloned into plasmid pcDNA3.0 in the MCS after CMV promoter just as shown in picture A, and then transfected into HEK293(PEI) and HepG2(Lipo2000). A GFP processed in the same way was used as a negative control to both indicate the transfection efficiency and normalize the death effect bring about by transfection process.Pictures were taken 48 hr after the transfection process.

To achieve a better resolution of the lethal effect, we tried two techniques with the 48hr cells. The first one is DAPI staining done in RIP1 and RIP3 groups. DAPI can specificly binds to DNA but is rejected by living cells to some extent. With DAPI staining, more cells were darkly dyed in the group of RIP1 and RIP3 compared to GFP control. Broken nuclei membrane can be noticed in cells transfected with RIP1 because DAPI blue spreaded all over cytopkasm The second one is flow cytometrycounting(FCM counting). Cells were stained by PI and then analysed by FCM counting. Dead cells binded more PI per cell in a similar reason to DAPI staining. These two techniques were complementary to each other in a certain degree, for DAPI is superior in morphological observation while FCM counting is good at counting.

Figure 1. Performance of different Suicide Genes candidates in causing apoptosis.(A).plasmids construction.(B)Apoptosis phenotype of Apoptin(VP3). The protein was expressed in fusion with a GFP. Compared with 0 hr , apoptosis rose 48 hr after transfection, and GFP vision indicated the transfection efficiency.(C,D) Wells transfected with RIP1\RIP3 both showed significant phenotype of apoptosis compared with GFP control, 48 hr after transfection. After DAPI staining, dead cell showed deep blue color and the phenotype was better visualized.(E)DAPI staining of RIP1, zoomed in vision.We can clearly observe boundary of cells which indicate a broken nucleus membrane and DNA spread all over the cells(F,G).PI staining and FCM data of our Suicide Gene.

The figure shows that Both RIP1 and RIP3 had a outstanding performance in mediating apoptosis. Although Apoptin also killed cell in the results, its performance wasn’t stable since we cannot reproduce the results with another construction( data not shown.) However, according to many paper, this is a powerful protein with a specificity in killing cancer cell, which persuade us to keep working with it in the latter experiments. Moreover, we also test Bax/Bax S184A with the same method. However, no significant phenotype could be observed(data not shown) and thus we abandoned them in our Suicide Gene list.

The PI staining and FCM data showed that the apoptosis rate of RIP1 and RIP3 were about 51.7% and 47.6%, which were significantly higher than the GFP control but rather lower than resent studies. However, this difference may be caused by the relatively low transfection efficiency in Heg G2. More details link to Modeling.

Sensor:miRNA binding site, the security gate for liver cell

Performance comparison between binding sites: CULT and Complete

Two kinds of binding site were taken into consideration just as mentioned in module Design. To make a comparison between their knockdown efficiency under different concentration of miR122, another orthogonal experiment was performed and two plasmids(fig2.A) were co-transfected by PEI into HEK293 cell line. Pictures were taken 24 hours after transfection.

Figure 2. Comparison of performance of different binding site in knocking down GFP expression. (A) Plasmids A/B/C/D produced mRNA of GFP with different binding site in its 3’ UTR. Plasmids E produced miR122. Plasmids used in this experiment had the problem of producing a background noise of GFP, which was solved later(see below).(B) Knockdown effect of different binding site under different miR122 level. The fields of vision were chosen so that they could well represent the average level of the wells.

The result shows that with the same copy number, complete binding site works better than CULT binding site, which fits the results of the 2010 Heiderburg iGEM team. What’s more, two copies binding site works better than single site, which encourage us to further build binding site of more copy number and employed more quantitating experiments.

Plasmid E used in the experiment produced background GFP, which was a problem neglected by us. After finding out the problem, we did quick-change to introduce a stop-codon into the CDS of GFP, which proves very efficient. The new miR122 expression plasmids were applied to other experiments just as mentioned. However, we found that although this did interfere our results to some degree, we could still gain insight into the problem so the data was kept.

Quantitative characterization and binding site modelling

In order to build a precisely controlled device, we decided to further characterized the complete binding site of different copy number and chose the most suitable one for pathway construction. To begin with we designed another orthogonal experiment crossing the two variables, binding site number N and MiR122 concentration M, with a more precise gradients for MiR122. But soon we found that it would be too labour-costing and decided that a better way was to replace it with 2 dosage-gradient experiments and a mathematic model. (fig.3)

Figure 3.Two strategies of characterizing miR122 binding site system.We adapt the latter strategy,making use of modeling principle,to simplify the work.

A.Dosage experiment testing performance of 2 copies complete binding site

Firstly we fixed the binding site number at 2 and set up a concentration gradient for miR122 expression plasmid (fig.7,A, plasmid CEP) to gain insight into its dosage effect. Plasmids A and B were co-transfected using PEI into HEK293 cell line. The gradient were set up from 0 to 0.5ug of plasmid per well. The experiment was done with 2 parallel groups of wells( that is, 6 wells as a group and there were 2 repeats) to leave samples for both Western-Blot and RT-qPCR(since both techniques require destruction of sample). To ensure the parallelity so that the two data can be employed to illustrate the problem together, we had repeated the experiment 2 times till we gained same fluorescence intensity with corresponding wells(data not shown).

Figure 4. Dosage effect of miR122 on 2 copy number binding site.(A)Plasmid CG2T produced GFP mRNA with a 2 copies Complete binding site in its 3’ UTR,while plasmid CEP produced miR122 without making fluorescence noise. (B)A gradient of total plasmid amount per well was set up as shown. A significant positive correlation between fluorescence knockdown efficiency and plasmid CEP concentration can be observed.(C) RT-qPCR showed that plasmid CEP successfully express miR122 in cells, the level of which was in a good linear correlation with the transfection concentration through linear-regression analysis(see Heptocyte). (D).Western-Blot showing the GFP level in each well.(E).GFP expression level derived by scanning densitometry,which is in good correlation with fluorescence observation.

The results showed a simple and elegant linear correlation between knockdown efficiency and miR122 concentration.What's more, we have planned to repeat the experiment and make a statistic data.

B.Performance of complete binding site of different copy number

Secondly we fixed the miR122 concentration at 0.75ug to ensure an optimal performance of each binding sites and let binding site number be the variable this time. Since we had already made sure of the relationship of miR122 level and plasmid concentration, RT-qPCR was no longer needed and so was the parallel groups.

Apart from the 2 and 4 copy binding site, we have also constructed a mixed-up plasmid with 2 complete binding site followed by 2 CULT binding site(2+2).

Figure 5. Comparison of knockdown efficiency of different binding site. (A).Plasmids construction for different binding site.(B).Knockdown effect of binding sites are significant in fluorescence vision.It can be observed that the 4*complete binding site has the most robust performance.(C).Western Blot of the experiment.The lanes has been cutted to omit some unrelated data.(D).Knockdown efficiency of the binding sites counted by data derived from Western Blot.

The results show that 4 copy binding site has the most brilliant knock-down efficiency, which is about under such a miR122 level, which conforms to the data given by 2010 Heiderburg using dual-fluorescent system. What’s more, in Part1 we have already proved that 2 copy binding site works better than 1 copy binding site. Finally we decided to first continue further construction of the pathway with the 2 copy binding site, considering that its moderate knockdown efficiency may well coordinate with the other two systems. And if the efficiency turns out improper, we can replace it with other candidates easily with our molecular designing.

C.Modeling

In order to better understand the quality of our miR122 binding site system, we have employed interpolation technique with Matlab to model its performance. The modeling can be seen at Modelling module in Results. Also, we have planned a more well-designed experiment to test and characterize all binding site(in fact, there are 8 of them, produced by permutation and combination of different numbers of complete and CULT). The data derived from that would better support the modeling work.

Figure 6. Modeling of MiR122 binding site system.

Switch:Tet-Off system,the controlling panel for iPSC

We also used eGFP to test the switching and driving performance of Tet-Off System, which would be a convenient controlling panel to switch pathway state between On and Off and thus provided protection for iPSC. We have collected and constructed three TRE elements of different generations.and thus made a comparison between them.

Figure 7. Performance of Tet-off system in switching between different state.Two plasmids(fig.7.A) were co-transfected into Bosc cell line using PEI. The wells with only plasmids B/C or D characterized the basal leaky expression of different pTRE elements, which can be viewed as the first Off-state. The well with both plasmids represents the On-state of the system, and the well further added with dox represents the second Off-state. Dox concentration was chosen according to protocols in order to give a best performance of the system and avoid hazard on cell growth.

The results showed that pTRE-Tight has the least leaky expression and a good switch response between On and Off state. However, in our experiments leaky expression was too high of pTRE2 and pTRE3G elements, and they cannot response to tTA and DOX properly(data not shown),which confused us a lot since according to protocol from Invitrogen, pTRE3G should have the best performance. We have repeated the experiments several times in troubleshooting, but gained no satisfactory results. So we decided to progress with pTRE-Tight in the first place and leave the other ones as alternative schemes.

In addition, we have received another version of Tet-off system in which the regulatory protein is fused by TetR and KRAB. According to our test result, the KRAB Tet-off system has the best fold increase but its leakage effect is higher the pTight. Maybe the leakage effect can be further eliminated by carefully select the optimal clone in stable cell lines. However, we abandon this system because KRAB theoretically does not work well in ES cells for our usage in iPSCs .

Figure 8. KRAB the new tet off system

Further test for parts combination

Since we have successfully proved that each part of our pathway worked well, we even wanted to further prove our device making use of the convenience of transient transfection, given that Lenti-Virus infection is too time-consuming. So we designed combinational experiments, centered on our protagonist, the Suicide Gene, to further confirmed the parts’ performance in working coordinately, including the test for combination of Suicide gene and tet-off system, the test for combination of Suicide Gene and miRNA binding site system and the test for the whole device. However, because of the time limited, we have to spend more energy on learning and researching the technology of stable cell lines establishment and assay, we finally did not choose to finish every experiments before Regional Jamboree.

Figure 9. Combinational test: Tet-Off system and Suicide Gene

Figure 10. Rescue experiment:miR122 binding site system and Suicide Gene

Figure 11. Orthogonal experiment of all three devices.

Lentivirus

Packaging:

We packaged two batches of lentivirus, one containing the upstream tTA plasmid, the other containing the downstream suicide gene and binding site plasmid.

Figure 12. The map of plasmids for lentivirus packaging.

The package of lentivirus vectors was done in HEK 293T cell lines in 10cm cell culture plates, along with two packaging plasmid: psPAX2 and pMD2G. Both of them were 3rd generation packaging plasmid. We then collected the CM supernatant after 48h and 72h, and transfered all of them into tubes.

Centrifugation:

Because these lentivirus vectors were supposed to infect not only Hep G2 cell lines but also mouse iPSCs, which was very difficult to infect, the requirement of the MOI (Multiplicity of Infection) was much higher than normal experiments, so before infecting cells, we ultracentrifugated these viral vectors.

Figure 13. The steps for lentivirus packaging and centrifugation.

Here is the photo of the 48hr culture medium with lentivirus.

Figure 14. The colors of culture medium with different lentivirus.

After successfully testing for each part, it's the right time to think about the assays in different period of cells.

Click the cells to see the results.

Sun Yat-Sen University, Guangzhou, China

Address: 135# Xingang Rd.(W.), Haizhu Guangzhou, P.R.China