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Team:Freiburg Bioware/Project/Targeting Fusion
2010-11-21T16:22:20Z
<p>Hanna: </p>
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<p class="MsoToc2"><span lang="DE"><a<br />
href="#_Toc275953546"><span lang="EN-US">Tumor<br />
Targeting by N-terminal Fusion to Virus Capsid<br />
</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953547"><span lang="EN-US">Design</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953548"><span lang="EN-US">VP2<br />
Fusion</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953549"><span lang="EN-US">VP1<br />
Insertion</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953550"><span lang="EN-US">Cloning<br />
Verified by Colony PCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953551"><span lang="EN-US">Fluorescence<br />
Analysis of Viral Stocks</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953552"><span lang="EN-US">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953553"><span lang="EN-US">Nuclear<br />
Localization by Fluorescence Microscopy</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953554"><span lang="EN-US">Transduction<br />
Efficacy by Flow Cytometry</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953555"><span lang="EN-US">Infectious<br />
Titer by qPCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953556"><span lang="EN-US">Killing<br />
cells: Time-Lapse</span></a></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h2><a name="_Toc275953546">Tumor Targeting by<br />
N-terminal Fusion to Virus Capsid<br />
</a></h2><br />
<h3><a name="_Toc275953547">Design</a></h3><br />
<h4><a name="_Toc275953548">VP2 Fusion</a></h4><br />
<p class="MsoNormal">The Freiburg2010 team fused motifs<br />
which are desired to be<br />
surface exposed to the N-terminus of VP2/3 open reading frame, avoiding<br />
steric<br />
hindrance by connecting them with linker. We ensured in frame coupling<br />
by<br />
designing or using the referring parts in the Freiburg RFC25 standard,<br />
thus<br />
creating fusion constructs via NgoMIV and AgeI. For retargeting AAV2<br />
virus<br />
particles towards EGFR over expressing cancer cells we investigated the<br />
Z<sub>EGFR:1907</sub><br />
Affibody and the E01 DARPin – in combination with VP2/3_587-KO (HSPG<br />
knock<br />
down) – as surface exposed motifs. We also created so called<br />
“all-in-one” virus<br />
particles by fusing VP2/3_His-Tag or VP2/3_BAP via Middle Linker to the<br />
Affibody. Purification or imaging approaches could be conducted by<br />
His-Tag or<br />
CFP motifs, respectively. All constructs were cloned downstream of the<br />
CMV<br />
promoter (Fig.1).</p><br />
<p class="MsoNormal"><b>Table 1: List of VP2 fusion<br />
parts</b></p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"></p><br />
<p class="MsoNormal"></p><br />
<br><br />
<p class="MsoNormal">&nbsp;&nbsp; <img<br />
alt="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"<br />
src="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"></p><br />
<p class="MsoNormal"><b>Figure 1: Cloning scheme of<br />
VP2 fusion</b></p><br />
<p class="MsoNormal">First of all we fused the Affibody<br />
ZEGFR:1907 to the Short-,<br />
Middle-, Long- or SEG Linker and cloned the resulting constructs<br />
downstream of the<br />
CMV promoter. Afterwards either unmodified VP2/3 or HSPG affinity knock<br />
down<br />
VP2/3 were fused to the different linkers. Further on CFP and His-Tag<br />
were<br />
coupled to VP2/3 by the Middle Linker (Fig. 2).</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p style="text-align: center;" class="MsoNormal"><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"<br />
src="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style=""><span style="">&nbsp; </span></span><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
2:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis.<br />
Fusion of VP2/3 to CMV_Affibody_Linker or CMV_CFP_MiddleLinker<br />
constructs.</span></p><br />
<p class="MsoNormal">We conducted three fragment ligations<br />
with the DARPin E01,<br />
MiddleLinker_VP2/3 or MiddleLinker_VP2/3(587KO) and pSB1C3_CMV plasmid<br />
(Fig.<br />
3).</p><br />
<p style="text-align: center;" class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
3:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis. <b style="">A)</b><br />
Three fragment ligation of DARPin<br />
fused to N-terminus of VP2/3. <b style="">B)</b><br />
Test digestion verified successful cloning.<o:p></o:p></span></p><br />
<p class="MsoNormal">For 100 % replacement of wildtype VP2<br />
the fusion plasmid was<br />
co-transfected to the Rep/Cap plasmid which contained a start codon<br />
mutation of<br />
VP2. Wildtype VP2 knock-out was achieved by site-directed mutagenesis<br />
(Fig. 4).<br />
</p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251659264; left: 0px; margin-left: 295px; margin-top: 26px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 4:<br />
Knock out of VP2</span></b> in the Cap coding region of<br />
Rep/Cap plasmid:<br />
Knock-out of start codon via site-directed mutagenesis (note the C in<br />
the sequence).</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h4><a name="_Toc275953549">VP1 Insertion</a></h4><br />
<p class="MsoNormal">We inserted three diffent motifs into<br />
the VP1 open reading<br />
frame. They were fused, together with the unique uspstream region of<br />
VP1<br />
(VP1up) and a nuclear localization sequence (NLS), according to the<br />
RFC25<br />
standard to VP2/3 (Fig. 2). Again, all of these parts were driven by<br />
CMV<br />
promoter.</p><br />
<p class="MsoNormal"><b>Table 2: List of VP1<br />
insertion parts</b></p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"<br />
src="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"><br />
</div><br />
<p class="MsoNormal"></p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
src="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
height="656" width="875"></p><br />
<p style="font-weight: bold;" class="MsoNormal">Figure<br />
5: Cloning scheme of VP1<br />
insertion</p><br />
<p class="MsoNormal">First VP1up-NLS was fused to the<br />
surface exposed motif –<br />
Affibody, His-Tag or mVenus – and the resulting constructs were cloned<br />
downstream of the CMV promoter. This was followed by either fusing<br />
unmodified<br />
VP2/3 or HSPG affinity knock down VP2/3 (Fig. 6) to the different kinds<br />
of<br />
motifs.</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="width: 450px; height: 362px;"<br />
src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"<br />
alt="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
6:<br />
Agarose Gel Electrophoresis.</span></b><span<br />
style="font-size: 10pt;"> Fusion of VP2/3(587KO) to<br />
pCMV_VP1up_NLS_ZEGFR:1907,<br />
pCMV_VP1up_NLS_6xHis or pCMV_VP1up_NLS_mVenus<o:p></o:p></span></p><br />
<p class="MsoNormal">We designed site-directed mutagenesis<br />
primer for the VP1<br />
start codon modification in the Rep/Cap plasmid in order to ensure 100<br />
%<br />
replacement with modified VP1 proteins (Fig. 7).</p><br />
<p style="text-align: center;" class="MsoNormal">&nbsp;<img<br />
style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"></p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251660288; left: 0px; margin-left: 252px; margin-top: 5px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 7:<br />
Knock out of VP1</span></b> in Cap coding region of Rep/Cap<br />
plasmid: Knock-out<br />
of start codon via site-directed mutagenesis.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953550">Cloning Verified by<br />
Colony PCR</a></h3><br />
<p class="MsoNormal">Big amounts of samples necessitated<br />
fast and exact analysis<br />
of cloning. For this purpose colony PCR was conducted. We verified<br />
successful<br />
VP2/3 fusion by using primer that exclusively bound to a specific<br />
location in<br />
the VP2/3 sequence (Fig. 8). In the case of successful ligation a 880<br />
bp<br />
fragment was amplified via Taq polymerase.</p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"><br />
</div><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 8:</span></b><br />
Primer Cap 3500 for and VP 4200 rev. Colony PCR using these primer<br />
results in a<br />
880 bp fragment</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 9 demonstrates a colony PCR<br />
example: VP2 fusion<br />
constructs containing VP2/3(587) sequence and the so called “All in<br />
One”<br />
constructs were cloned into pSB1C3_CMV. Successful cloning could be<br />
verified<br />
(positive control: pAAV_RC; negative control: pSB1C3_lITR).</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
9: Colony<br />
PCR</span></b><span style="font-size: 10pt;"><br />
of VP2<br />
N-terminal fusion (Affibody_Linker, CFP_MiddleLinker,<br />
His-Tag_MiddleLinker:<br />
samples 1 - 6) and “All-in-One” constructs (samples 7 – 10).<o:p></o:p></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953551">Fluorescence Analysis of<br />
Viral Stocks</a></h3><br />
<p class="MsoNormal">We wanted to develop a protocol for<br />
harvesting as much<br />
viruses as possible. For this purpose we took advantage of viruses<br />
containing<br />
mVenus fused to VP1. In order to find out whether most viruses were<br />
located in<br />
the cell pellet, cell suspensions were centrifuged at 10.000 g or<br />
20.000 g.<br />
Afterwards mVenus fluorescence was measured at 526 nm. The resulting<br />
values were<br />
compared to cell culture supernatant, 20.000 g centrifuged supernatant<br />
and<br />
resuspended cell pellet (Fig. 10).</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in; width: 700px; height: 525px;"<br />
alt="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"<br />
src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"><br />
</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
10: Fluorescence signal of mVenus at 526 nm</span></b>,<br />
inserted into the virus<br />
capsid. Transduced cells were resuspended, pelleted or supernatant was<br />
taken in<br />
order to determine the location of most viruses. Control: DMEM.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953552">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</a></h3><br />
<p class="MsoNormal">For further analysis, viral stocks<br />
originating from cell<br />
culture supernatant needed to be concentrated. Therefore<br />
ultrafiltration with<br />
protein concentrators (Sartorius VivaSpin 20; 20.000Da MWCO) was<br />
conducted.<br />
Figure 11 shows fluorescence microscopy image of an aliquot from<br />
ultrafiltrated<br />
cell culture supernatant / cell lysate containing<br />
VP1up_NLS_mVenus_VP2/3<br />
viruses.</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"<br />
src="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
11: Fluorescence Microscopy</span></b>. A) Brightfield<br />
picture of an aliquot of<br />
concentrated VP1up_NLS_mVenus_VP2/3 containing virus stock. B) At 505<br />
nm<br />
excitation mVenus fluorescence of the virus particles could be<br />
detected. C)<br />
Merged image</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953553">Nuclear Localization by<br />
Fluorescence Microscopy</a></h3><br />
<p class="MsoNormal">AAV-293 cells were transfected with a<br />
50:50 ratio of the<br />
Rep/Cap(VP1KO) to the CMV_VP1_NLS_mVenus_VP2/3 plasmid. We packaged<br />
mCherry,<br />
driven by the CMV promoter, into the virus capsids and followed protein<br />
expression<br />
via fluorescence microscopy. 30 hours post transfection mCherry<br />
fluorescence<br />
was detectable in the whole cytosol of the successfully transfected<br />
cells,<br />
demonstrating that DNA located between the AAV2 ITRs is already<br />
transcribed in<br />
the producer cell line. In contrast to that mVenus fluorescence signal<br />
could be<br />
observed only in the nuclei (Fig. 12). This indicated that the nuclear<br />
localization sequence targets the single VP1_NLS_mVenus_VP2/3 proteins<br />
efficiently to the cell nucleus, where assembly and packaging of the<br />
virus<br />
particles takes place.&nbsp; </p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
src="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
height="656" width="875"></p><br />
<p class="MsoNormal" style="line-height: normal;">&nbsp;<br><br />
<b style=""><span style="font-size: 10pt;">Figure<br />
12:<br />
Fluorescence Microscopy. A) </span></b><span<br />
style="font-size: 10pt;">Brightfiled picture. <b style="">B)</b><br />
Excitation at 555 nm showed mCherry signal in the cytosol. <b<br />
style="">C)</b> Excitation at 505 nm revealed mVenus<br />
fluorescence in the nuclei, indicating functionality of the nuclear<br />
localization sequence inserted – together with mVenus – into VP1. <b<br />
style="">D)</b> Merged image.</span></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style="font-size: 10pt;"><o:p></o:p></span></p><br />
<h3><a name="_Toc275953554">Transduction Efficacy by<br />
Flow Cytometry</a></h3><br />
<p class="MsoNormal">For determination of transduction<br />
efficacy flow cytometry<br />
analysis was conducted. The Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
was fused with SEG-, middle- and long<br />
linker to the VP2/3 open reading frame (ORF) in order to investigate<br />
differences in infection efficacy due to different linker lengths.<br />
250.000<br />
AAV-293 cells were transfected with 1 µg total DNA. Different ratios of<br />
VP2<br />
fusion constructs in respect to the Rep/Cap plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and two different cell<br />
lines,<br />
HT1080 and A431, were transduced with 1 mL virus stock. By<br />
encapsulating mVenus<br />
coding sequence, the amount of transduced cells could be determined via<br />
flow<br />
cytometry.</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"<br />
src="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 13: Flow<br />
cytometry.</span></b><span style=""> Test of<br />
transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">Middlelinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p style="text-align: center;" class="MsoNormal"> <img<br />
alt="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 14: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with<br />
HT1080 and A431 cells by detecting mVenus expression from <b>Z<sub>EGFR:1907</sub>_SEG_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p class="MsoNormal"<br />
style="line-height: normal; text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"<br />
src="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 15: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">LongLinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span><b><span style="font-size: 12pt;"><o:p></o:p></span></b></p><br />
<span style=""><o:p></o:p></span><br />
<p class="MsoNormal">Additionally viruses containing a<br />
His-Tag or CFP, fused via<br />
Middle Linker to the VP2/3 ORF, were analyzed. Figure 16 overviews<br />
transduction<br />
efficacy of all constructs, transfected in a 10:90, 25:75 and 50:50<br />
ratio in<br />
respect to Rep/Cap plasmid.&nbsp;&nbsp; </p><br />
<p style="text-align: center;" class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"<br />
src="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"></p><br />
<p class="MsoNormal"></p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"><br><br />
</div><br />
<p class="MsoNormal"><b>Figure 16: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 and A431 cells, infected with virus<br />
particles<br />
consisting of different ratios of VP2 fusion constructs in respect to<br />
Rep/Cap<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Transduction of HT1080 cells revealed<br />
that all viral<br />
particles regardless of which motifs inserted into the capsids remained<br />
infectious with efficacies up to 85 %. In general the amount of mVenus<br />
positive<br />
cells decreased only slightly when harboring more modified VP2<br />
subunits. This<br />
indicates that larger peptides could be inserted into the AAV2 capsids<br />
without<br />
affecting virus assembly and packaging. A431 cells, which overexpress<br />
EGF<br />
receptor, were generally transduced with reduced efficacy. </p><br />
<p class="MsoNormal">Further on the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
or mVenus were inserted into the VP1<br />
ORF together with a nuclear localization signal. 250.000 AAV-293 cells<br />
were<br />
transfected with 1 µg total DNA and different ratios of VP1 insertion<br />
constructs in respect to the Rep/Cap(VP1KO) plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and HT1080 and A431<br />
cells were<br />
transduced. 48 hours later the number of transduced, or to be precise<br />
mVenus<br />
positive, cells was determined via flow cytometry (Fig. 17 a, b).</p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"><br />
</div><br />
<p class="MsoNormal"><b>Figure 17 a: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 cells, infected with virus particles<br />
containing different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO) plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"></p><br />
<p class="MsoNormal"><b>Figure 17 b: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive A431 cells, infected with virus particles<br />
containing<br />
different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO)<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Again results revealed that all virus<br />
particles remained<br />
infectious, regardless of whether inserting the Affibody or mVenus.<br />
This<br />
indicated that VP1 tolerated larger peptides inserted downstream of its<br />
unique<br />
N-terminal region and that this modification still allowed virus<br />
assembly and<br />
packaging.</p><br />
<h3><a name="_Toc275953555">Infectious Titer by qPCR</a><br />
</h3><br />
<p class="MsoNormal">We transfected 250.000 AAV-293 cells<br />
with 1 µg of total DNA<br />
composed of equal amounts of Rep/Cap, pHelper and vector plasmid. VP2<br />
fusion or<br />
VP1 insertion plasmids were co-transfected with two different ratios in<br />
respect<br />
to Rep/Cap(VP1KO) or Rep/Cap(VP2KO):&nbsp; 25&nbsp;% VP2 fusion<br />
or VP1 insertion<br />
proteins allow better assembly and packaging of the virus particles,<br />
compared<br />
to 50 % VP2 fusion or VP1 insertion proteins which increase the chance<br />
of&nbsp;<br />
integration into the capsids. </p><br />
<p class="MsoNormal">We created viruses containing the Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
Affibody fused<br />
to VP2 or inserted into VP1 and the DARPin E01 fused to VP2. These<br />
types of<br />
AAV2 particles were produced in two versions: With or without HSPG<br />
binding<br />
affinity knock down (587KO). </p><br />
<p class="MsoNormal">Viruses were harvested three days<br />
post transfection. The genomic<br />
titer was determined via qPCR by amplification of a specific sequence<br />
located<br />
in the CMV promoter of the vector plasmid (Table 3).</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Table 3:<br />
Quantitative Real-Time PCR.</span></b> Determination of<br />
genomic titer. Data<br />
were corrected for negative control value.</p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border: medium none ; width: 450.2pt; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"<br />
width="600"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Co-transfected<br />
Construct</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Ratio</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Genomic<br />
Titer /1ml</span></b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Corrected<br />
For Negative Control</span></b></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,20E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,39E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,17E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,44E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,63E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,67E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,37E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,38E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,52E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,50E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6,98E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,30E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,25E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,65E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">4,36E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,93E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,00E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,99E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,55E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,39E+08</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We investigated transduction of<br />
different cell lines. For<br />
this purpose 100.000 HT1080, HeLa or A431 cells were seeded and<br />
transduced with<br />
50 µL virus stock and harvested 24 hours later.&nbsp; Infectious<br />
titers were<br />
determined via qPCR and normalized to the genomic titers (Fig. 17-19).</p><br />
<p class="MsoNormal">Figure 18 shows infection efficacy of<br />
DARPin exposing<br />
viruses. Transduction of HT1080 cells was almost not affected as long<br />
as<br />
binding to HSPG was not knocked down. HeLa cells were also infected<br />
less<br />
efficient compared to the controls. However, A431 cells which<br />
overexpress EGFR<br />
were not infected by the controls. Transduction is rescued by<br />
integration of<br />
the DARPin into the virus capsid. By additionally knocking down the<br />
HSPG<br />
binding affinity these cells are transduced 10 times better, reaching<br />
wild type<br />
capsid HT1080 infection efficacy. These results indicated that specific<br />
re-targeting of AAV2 virus particles towards EGFR over expressing tumor<br />
cells<br />
was achieved by N-terminal fusion of targeting motifs to VP2.<br><br />
<br><br />
<br><br />
</p><br />
<div style="text-align: center;"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
height="656" width="875"></div><br />
<p class="MsoNormal"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 18:<br />
DARPin E01 VP2 Fusion.</span></b> Infectious titers were<br />
determined with or<br />
without HSPG knock down for HT1080, HeLa and A431 cells.<br />
Control:Rep/Cap<br />
plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We additionally obtained similar<br />
results for Affibody<br />
exposing viruses: A431 cell transduction could also be rescued by<br />
capsid<br />
integration of the Affibody. Again infection efficacy was increased by<br />
knocking<br />
down the natural tropism of the AAV2 viruses towards HSPG (Fig. 19).<br />
These data<br />
emphasized the functionality of the VP2 fusion constructs for<br />
specifically<br />
targeting tumor cells for therapeutic or imaging<br />
applications.&nbsp; </p><br />
<p class="MsoNormal"></p><br />
<div style="text-align: center;"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
height="656" width="875"><br><br />
</div><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
19:<br />
Affibody Z<sub>EGFR:1907</sub> VP2 Fusion.</span></b><br />
Infectious titers were<br />
determined with or without HSPG knock down for HT1080, HeLa and A431<br />
cells.<br />
Control:Rep/Cap plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 20 repesents the infectivity<br />
of viruses containing<br />
VP1 inserted Affibody molecules in respect to three different cell<br />
types<br />
(HT1080, HeLa, A431). The data clearly shows that virus capsids<br />
assembled and<br />
remained infectious. These AAV2 particles also featured specific<br />
binding<br />
properties to A431 cells, validating the functionality of&nbsp; the<br />
VP1 insertion<br />
strategy. The two outlier (HeLa VP1up_NLS_Affibody_VP2/3 25:75<br />
&amp; HT1080<br />
VP1up_NLS_Affibody_VP2/3(587KO) 50:50) should be left unconsidered. </p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
height="656" width="875"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 20:<br />
Affibody Z<sub>EGFR:1907</sub> VP1 Insertion.</span></b><br />
Infectious titers were<br />
determined for 25:75 and 50:50 transfection ratios with or without HSPG<br />
knock<br />
down for HT1080, HeLa and A431 cells. Control:Rep/Cap plasmid with and<br />
without<br />
HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953556">Killing cells: Time-Lapse</a></h3><br />
<p class="MsoNormal">HT1080 and A431 cells were transduced<br />
with so called<br />
“All-in-One” viruses containing the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
fused to VP2/3(587KO_His-Tag) and<br />
packaged with the guanylate kinase fused to the thymidine kinase coding<br />
sequence (GMK-TK). A time series of pictures was started directly after<br />
adding<br />
20 µM Ganciclovir (Fig.21).</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
a: Time-lapse</span></b>. HT1080 (control) cells transduced<br />
with GMK-TK<br />
packaged viruses and treated with 20 µM Ganciclovir A) 0 hours, B) 7<br />
hours, C)<br />
15 hours and D) 23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"<br />
src="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
b: Time-lapse</span></b>. A431 cells transduced with GMK-TK<br />
packaged viruses<br />
and treated with 20 µM Ganciclovir A) 0 hours, B) 7 hours, C) 15 hours<br />
and D)<br />
23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Virus and Ganciclovir treatment only<br />
slightly affect the<br />
morphology of HT1080 cells. After 23&nbsp;hours of incubation in<br />
20µM<br />
Ganciclovir the cells had almost the same appearance as at time point<br />
zero<br />
(Fig. 21 a). In comparison to that A431 epidermoid carcinoma cells were<br />
efficiently killed after transduction and Ganciclovir add-on: After 23<br />
hours<br />
nearly all cells were lysed (Fig. 21 b). These results clearly<br />
demonstrate that<br />
we were able to specifically target EGFR over-expressing tumor cells<br />
via capsid<br />
integrated Affibody and that these transduced cells were efficiently<br />
killed by<br />
expressing the GMK-TK which converted prodrug Ganciclovir into its<br />
cell-toxic monophosphate.<br />
</p><br />
<br />
<br/><br/><br/><br />
<br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
</html></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Team/Team_2010
Team:Freiburg Bioware/Team/Team 2010
2010-10-27T22:54:51Z
<p>Hanna: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}} <br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>The iGEM 2010 Team Freiburg</h1><br />
<br />
<img src="https://static.igem.org/mediawiki/2010/6/61/Freiburg10_Team_picture.png"<br />
style="width: 600px; height: auto; margin: 20px 0px 20px 170px;"><br />
<br />
<p style="text-align: justify;"><br />
We are a team<br />
of students<br />
consisting of 15 members. Among, there are 13 biology students<br />
specialized in<br />
various topics such as Biochemistry, Genetics, Microbiology, Plant<br />
Biotechnology, Immunology and of course Synthetic Biology. Furthermore,<br />
our<br />
team has one student of physics and one student of informatics.<br />
Combining these<br />
abilities we handled the challenges associated with our project and<br />
realized it<br />
hand in hand. Over the past summer, the team of biology students worked<br />
in<br />
groups and was greatly supported by our instructors and supervisor.<br />
Each group consisted<br />
of 2-3 students and was specialized either on DNA cloning or on cell<br />
culture.<br />
These main topics were again divided into smaller ones, like for<br />
example<br />
‘N-terminal fusion’ or ‘loop modifications’ of our virus. As our<br />
labjournal<br />
editings reached unlimited dimensions, we realized that we need further<br />
help. Luckily<br />
our Physics and Computer sciences students reinforced our team and<br />
designed the<br />
wiki and created mathematical models. <br><br />
Besides the<br />
daily<br />
laboratory work, we all spent some free time together, watching movies<br />
cooking,<br />
and having fun together. One of the funniest events was our canoe trip,<br />
sunny<br />
weather but however, wet conditions.<br><br />
For pictures see: [Link]</p><br />
<p style="text-align: justify;"><br><br />
</p><br />
<h1>Team members</h1><br />
<h2>Instructors</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/86/Freiburg10_Team_Kristian_Mueller.png"<br />
alt="Kristian Müller" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Kristian Müller</h5><br />
<p><br />
Kristian is our supervisor, he coordinates and reinforces us in our<br />
efforts and plans.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Katja_Arndt.png"<br />
alt="Katja Arndt" width="368" style="margin-right: 15px;"/><br />
<br />
<div><h5>Katja Arndt</h5><br />
<p><br />
<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left"<br />
src="https://static.igem.org/mediawiki/2010/7/71/Freiburg10_Team_Sven_Hagen.png"<br />
alt="Sven Hagen" width="368" style="margin-right: 15px;"/><br />
<div><h5>Sven Hagen</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Tobi, of course. Thank you for everything.<br />
<br><br />
<br><br />
Citation: Start by doing what's necessary; then do what's possible; and suddenly you are doing the impossible. (St. Francis of Assisi 1181/1182 – 1226)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><img class="left"<br />
src="https://static.igem.org/mediawiki/2010/3/31/Freiburg10_Team_Tobias_Baumann.png"<br />
alt="Tobias Baumann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Tobias Baumann</h5><br />
<p><br />
To tell the truth, he is the best instructor a team can have. Stop!<br />
Besides Sven, of course. Thank you for everything.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<br />
<h2>Student Members</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/55/Freiburg10_Team_Achim_Mall_.png"<br />
alt="Achim Mall" width="368" style="margin-right: 15px;"/><br />
<div><h5>Achim Mall</h5><br />
<p><br />
Achim is surrounded by an aura of silence. Pipete more than 100 qPCRs?<br />
No problem! Keep cool and then let’s go.<br><br />
<br><br />
Citation: I love deadlines. I like the whooshing sound they make as<br />
they fly by. (Douglas Adams)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
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<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/e/e3/Freiburg10_Team_Adrian_Fischer.png"<br />
alt="Adrian Fischer" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Adrian Fischer</h5><br />
<p><br />
This is Adrian, he is not only one of the best in cell culture, but he<br />
brought music from all centuries (preferring the 90’s!) into our lives.<br />
<br><br />
<br><br />
Citation: “You have to make the good out of the bad, because that is<br />
all you have got to make it out of." Robert Penn Warren<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6d/Freiburg10_Team_Anissa_Bender.png"<br />
alt="Anissa Bender" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anissa Bender</h5><br />
<p><br />
Anissa has skills in cloning and cell culture works, both without any<br />
difficulty. And if you need somebody to be really honest, she is the<br />
right person.<br><br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/87/Freiburg10_Team_Anna_Oschowitzer.png"<br />
alt="Anna Oschowitzer" width="368" style="margin-right: 15px;"/><br />
<div><h5>Anna Oschowitzer</h5><br />
<p><br />
She`s the heart and soul of the lab. Always good-humored and poised to<br />
do that needs to be done. She has skills in cloning and PCR and did a<br />
good job in winning sponsors.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_Team_Bea_Kaufmann.png"<br />
alt="Bea Kaufmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Bea Kaufmann</h5><br />
<p><br />
Our ‘laboratory smurf’ is an organizing talent and takes care of the<br />
whole project and its single members.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/c/c3/Freiburg10_Team_Christian_Weing%C3%A4rtner.png"<br />
alt="Christian Weingärtner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Christian Weingärtner</h5><br />
<p><br />
He is the real midiprep master, the nanodrop is overstrained when<br />
measuring these enormous concentrations.<br />
<br><br />
<br><br />
Citation: "All the world's a stage" William Shakespeare<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/7/7e/Freiburg10_Team_Hanna_Wagner.png"<br />
alt="Hanna Wagner" width="368" style="margin-right: 15px;"/><br />
<div><h5>Hanna Wagner</h5><br />
<p><br />
The ITRs were nearly incomprehensible, until now! Here we have somebody<br />
with the fanciest methods and cloning strategies.<br/><br />
<br/><br />
Citation: I believe there is no philosophical high-road in science, with epistemological signposts. No, we are in a jungle and find our way by trial and error, building our road behind us as we proceed.<br />
(Max Born) <br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
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<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/63/Freiburg10_Team_Jessica_G%C3%BCnzle.png"<br />
alt="Jessica Günzle" width="368" style="margin-right: 15px;"/><br />
<div><h5><br />
Jessica Günzle</h5><br />
<p><br />
Jessica is a multitalent. Planning our iGEM cajak tour, our trip and<br />
flight to Boston and working in the lab at the same time. That is<br />
great, man! <br><br />
<br><br />
Citation: Equipped with his five senses, man explores the universe<br />
around him and calls the adventure Science. (Edwin Powell Hubble)<br />
</p><br />
</div><br />
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</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/84/Freiburg10_Team_Kerstin_Klingele.png"<br />
alt="Kerstin Klingele" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kerstin Klingele</h5><br />
<p><br />
Our sweet little girl does great work in cell culture and using flow<br />
cytometry. You can be sure with every business, it is all in good<br />
hands.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/d/d7/Freiburg10_Team_Kira_Meyerovich.png"<br />
alt="Kira Meyerovich" width="368" style="margin-right: 15px;"/><br />
<div><h5>Kira Meyerovich</h5><br />
<p><br />
Laboratory experience is greatly available. And a very smart way of<br />
working, too.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/6/6b/Freiburg10_Team_Max_Schelker.png"<br />
alt="Max Schelker" width="368" style="margin-right: 15px;"/><br />
<div><h5>Max Schelker</h5><br />
<p><br />
Max is our physicist and a specialist in Systems Biology, he really<br />
knows how to handle data-based mathematical modeling. <br><br />
<br><br />
Citation: "Do not worry about your difficulties in Mathematics. I can<br />
assure you<br />
mine are still greater." (Albert Einstein)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/b7/Freiburg10_Team_Stefan_Bergmann.png"<br />
alt="Stefan Bergmann" width="368" style="margin-right: 15px;"/><br />
<div><h5>Stefan Bergmann</h5><br />
<p><br />
If you think there is no helping hand, you’re wrong. Stefan is<br />
everywhere and has got the plans in mind. <br><br />
<br><br />
Citation: "We are just an advanced breed of monkeys on a minor planet<br />
of a very average star. But we can understand the Universe. That makes<br />
us something very special."<br />
(S. Hawking)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/5/57/Freiburg10_Team_Volker_Morath.png"<br />
alt="Volker Morath" width="368" style="margin-right: 15px;"/><br />
<div><h5>Volker Morath</h5><br />
<p><br />
Guess who knows how to build a virus in his own basement. And if you<br />
need a straight opinion, go for Volker. <br><br />
<br><br />
Citation: Quand tu veux construire un bateau, ne commence pas par<br />
rassembler du bois, couper des planches et distribuer du travail, mais<br />
reveille au sein des hommes le desir de la mer grande et large.<br />
(Antoine de Saint-Exupéry)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/4/4e/Freiburg10_Team_Patrick_Schindler.png"<br />
alt="Patrick Schindler" width="368" style="margin-right: 15px;"/><br />
<div><h5>Patrick Schindler</h5><br />
<p><br />
He is a hard worker, whether there is something to do in the<br />
laboratory, or to eat meals for a whole team. <br><br />
<br><br />
Citation: If You Want Peace, prepare for war. (Publius Flavius Vegetius<br />
Renatus)<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/8/8f/Freiburg10_Team_Timon.png"<br />
alt="Timon Schober" width="368" style="margin-right: 15px;"/><br />
<div><h5>Timon Schober</h5><br />
<p><br />
Do you see this great wiki? Yes, this is the work of our great web<br />
programmer and computer scientist Timon.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<h2>Friends</h2><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/9/90/Freiburg10_Team_Fabian_Haas.png"<br />
alt="Fabian Haas" width="184" style="margin-right: 15px; margin-left:183px;"/><br />
<div><h5>Fabian Haas</h5><br />
<p></p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
<div class="box box_full"><br />
<img class="left" src="https://static.igem.org/mediawiki/2010/b/be/Freiburg10_Team_Jo_Zang.png"<br />
alt="" width="368" style="margin-right: 15px;"/><br />
<div><h5>Johannes Zang</h5><br />
<p><br />
He takes the best pictures ever and knows how to fight with photoshop.<br />
</p><br />
</div><br />
<div class="clearfix"></div><br />
</div><br />
<br />
</html><br />
{{:Team:Freiburg_Bioware/Footer}}</div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal
Team:Freiburg Bioware/NoteBook/Labjournal
2010-10-27T19:15:28Z
<p>Hanna: </p>
<hr />
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<!---Boxes for the months---><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March"><br />
<h2 style="margin-left: 5px;">March</h2><br />
<p class="boxes_labjournal_day">Labday 1</p><br />
<img src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg_10_labjournal_march_v1.JPG" class="image_labjournal" title="Our project starts with first sequence analysis."/><br />
<p class="boxes_labjournal_text">...watching gene sequences...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April"><br />
<h2 style="margin-left: 5px;">April</h2><br />
<p class="boxes_labjournal_day">Labday 2- 5</p><br />
<img src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites: <br />
Vector plasmid; Thymidine kinase ;Cytosindeaminase" /><br />
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May"><br />
<h2 style="margin-left: 5px;">May</h2><br />
<p class="boxes_labjournal_day">Labday 6- 17</p><br />
<img src="https://static.igem.org/mediawiki/2010/0/0f/Freiburg_10_labjournal_may_v1.JPG" class="image_labjournal" title="We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt." /><br />
<p class="boxes_labjournal_text">...theoretical cloning...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June"><br />
<h2 style="margin-left: 5px;">June</h2><br />
<p class="boxes_labjournal_day">Labday 18- 45</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title="<br />
We conducted our first site-directed mutagenesis. First cell culture steps: Splitting and seeding of AAV 293 and HT1080 cells; Calcium phosphate transfection; Transduction with virus particles containing YFP encoding sequence; Microscopy: Fluorescent transduced HT1080 cells. We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. Theoretical studies of the AAV structure: Impressions" /><br />
<p class="boxes_labjournal_text">...first transduced cells...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July"><br />
<h2 style="margin-left: 5px;">July</h2><br />
<p class="boxes_labjournal_day">Labday 46- 75</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid.<br />
For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI. BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. Further on we received first quantitative transduction results via flow cytometry. mGMK_TK30 was successfully cloned into the vector plasmid." /><br />
<p class="boxes_labjournal_text">...organizing the lab...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August"><br />
<h2 style="margin-left: 5px;">August I</h2><br />
<p class="boxes_labjournal_day">Labday 76- 92</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/Freiburg_10_labjournal_august1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...cloning parts...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2"><br />
<h2 style="margin-left: 5px;">August II</h2><br />
<p class="boxes_labjournal_day">Labday 93- 106</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg_10_labjournal_august2_v1.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...assembled vector plasmid is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September"><br />
<h2 style="margin-left: 5px;">September I</h2><br />
<p class="boxes_labjournal_day">Labday 107- 123</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/25/Freiburg_10_labjournal_september1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...simply producing BioBricks...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2"><br />
<h2 style="margin-left: 5px;">September II</h2><br />
<p class="boxes_labjournal_day">Labday 124- 135</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...21x mutated RepCap is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October"><br />
<h2 style="margin-left: 5px;">October I</h2><br />
<p class="boxes_labjournal_day">Labday 136- 149</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/e0/Freiburg_10_labjournal_october_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...testing BioBricks by virus production...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2"><br />
<h2 style="margin-left: 5px;">October II</h2><br />
<p class="boxes_labjournal_day">Labday 150- 166</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Capsid modification works...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November"><br />
<h2 style="margin-left: 5px;">November</h2><br />
<p class="boxes_labjournal_day">Labday 166- 170</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Jemboree!!!...</p><br />
</a><br />
</div><br />
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March (labday 1)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April (labday 2 - 5)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May (labday 6 - 17)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June">June (labday 18 - 45)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July">July (labday 46 - 75)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August part 1 (labday 76 - 92)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2">August part 2 (labday 93 - 106)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November (labday 167 - 170 )</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li><br />
</ul><br><br><br><br><br><br><br><br><br><br><br />
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Hanna
http://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal
Team:Freiburg Bioware/NoteBook/Labjournal
2010-10-27T19:14:55Z
<p>Hanna: </p>
<hr />
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<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March"><br />
<h2 style="margin-left: 5px;">March</h2><br />
<p class="boxes_labjournal_day">Labday 1</p><br />
<img src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg_10_labjournal_march_v1.JPG" class="image_labjournal" title="Our project starts with first sequence analysis."/><br />
<p class="boxes_labjournal_text">...watching gene sequences...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April"><br />
<h2 style="margin-left: 5px;">April</h2><br />
<p class="boxes_labjournal_day">Labday 2- 5</p><br />
<img src="https://static.igem.org/mediawiki/2010/f/fc/Freiburg_10_labjournal_april_v1.JPG" class="image_labjournal" title="Scanning for iGEM restriction sites: <br />
Vector plasmid; Thymidine kinase ;Cytosindeaminase" /><br />
<p class="boxes_labjournal_text">...ordering AAV-2 Helper-free System...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May"><br />
<h2 style="margin-left: 5px;">May</h2><br />
<p class="boxes_labjournal_day">Labday 6- 17</p><br />
<img src="https://static.igem.org/mediawiki/2010/0/0f/Freiburg_10_labjournal_may_v1.JPG" class="image_labjournal" title="We took a closer look to the theoretical DNA- and amino acid sequence of the ITRs, questioned the role of the β-globin intron, had a closer look to the CMV promoter and performed our first cloning attempt." /><br />
<p class="boxes_labjournal_text">...theoretical cloning...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June"><br />
<h2 style="margin-left: 5px;">June</h2><br />
<p class="boxes_labjournal_day">Labday 18- 45</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/37/Freiburg_10_labjournal_june_v1.jpg" class="image_labjournal" title="<br />
We conducted our first site-directed mutagenesis. First cell culture steps: Splitting and seeding of AAV 293 and HT1080 cells; Calcium phosphate transfection; Transduction with virus particles containing YFP encoding sequence; Microscopy: Fluorescent transduced HT1080 cells. We planned to determine infectious virus titer via quantitative real-time PCR. For this purpose primers were designed, transduced HT1080 cells were harvested and prepared. Theoretical studies of the AAV structure: Impressions" /><br />
<p class="boxes_labjournal_text">...first transduced cells...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July"><br />
<h2 style="margin-left: 5px;">July</h2><br />
<p class="boxes_labjournal_day">Labday 46- 75</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8d/Freiburg_10_labjournal_july_v1.JPG" class="image_labjournal" title="We developed a plan for modifying the surface exposed loops of the virus capsid.<br />
For this purpose not only 5 iGEM restriction sites, but also the so called ViralBrick restriction sites located in the Rep/Cap sequence needed to be deleted: BamHI, SalI. BioBrick production of the inverted terminal repeats (ITRs) turned out to be difficult. Therefore the alternative “ITR fancy method” was developed. We successfully produced AAV2 left and right ITR fused to the RFC10 prefix. Further on we received first quantitative transduction results via flow cytometry. mGMK_TK30 was successfully cloned into the vector plasmid." /><br />
<p class="boxes_labjournal_text">...organizing the lab...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August"><br />
<h2 style="margin-left: 5px;">August I</h2><br />
<p class="boxes_labjournal_day">Labday 76- 92</p><br />
<img src="https://static.igem.org/mediawiki/2010/8/8b/Freiburg_10_labjournal_august1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...cloning parts...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2"><br />
<h2 style="margin-left: 5px;">August II</h2><br />
<p class="boxes_labjournal_day">Labday 93- 106</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg_10_labjournal_august2_v1.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...assembled vector plasmid is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September"><br />
<h2 style="margin-left: 5px;">September I</h2><br />
<p class="boxes_labjournal_day">Labday 107- 123</p><br />
<img src="https://static.igem.org/mediawiki/2010/2/25/Freiburg_10_labjournal_september1_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...simply producing BioBricks...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2"><br />
<h2 style="margin-left: 5px;">September II</h2><br />
<p class="boxes_labjournal_day">Labday 124- 135</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...21x mutated RepCap is working...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October"><br />
<h2 style="margin-left: 5px;">October I</h2><br />
<p class="boxes_labjournal_day">Labday 136- 149</p><br />
<img src="https://static.igem.org/mediawiki/2010/e/e0/Freiburg_10_labjournal_october_v1.JPG" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...testing BioBricks by virus production...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2"><br />
<h2 style="margin-left: 5px;">October II</h2><br />
<p class="boxes_labjournal_day">Labday 150- 166</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Capsid modification works...</p><br />
</a><br />
</div><br />
<br />
<div class="box_labjournal_195"><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November"><br />
<h2 style="margin-left: 5px;">November</h2><br />
<p class="boxes_labjournal_day">Labday 166- 170</p><br />
<img src="https://static.igem.org/mediawiki/2010/3/35/Freiburg_10_BioBrick_icon.png" class="image_labjournal" /><br />
<p class="boxes_labjournal_text">...Jemboree!!!...</p><br />
</a><br />
</div><br />
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https://2010.igem.org/Team:Freiburg_Bioware/NoteBook => Back to Notebook overview]<br />
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<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/March">March (labday 1)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/April">April (labday 2 - 5)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/May">May (labday 6 - 17)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/June">June (labday 18 - 45)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/July">July (labday 46 - 75)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August">August part 1 (labday 76 - 92)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/August2">August part 2 (labday 93 - 106)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September">September part 1 (labday 107 - 123)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/September2">September part 2 (labday 124 - 135)</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October">October part 1 (labday 136 - 149 )</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/October2">October part 2 (labday 150 - 166 )</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/November">November (labday 167 - 170 )</a></li><br />
<li><a href="https://2010.igem.org/Team:Freiburg_Bioware/NoteBook/Labjournal/Cellculture">Cellculture</a></li><br />
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Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Project/Results
Team:Freiburg Bioware/Project/Results
2010-10-27T15:34:37Z
<p>Hanna: </p>
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<h1>Results</h1><br />
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<a name="highlights"><h2>Highlights</h2></a><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
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<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
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<a name="modularization"><h2>Modularization</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Mod Vector Plasmid</a><br />
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
</p><br />
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<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
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<a name="targeting"><h2>Targeting</h2></a><br />
<p>One aim of our research is on the one hand to knock down the natural tropism of the Adeno-associates virus particles and on the other hand to specifically target tumor cells. This is achieved by genetic engineering of the virus surface. For this purpose, two different strategies were developed, including <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/insertion of motifs into surface-exposed loops">Targeting via Loops</a> or <a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion">fusion to the N-terminus of the viral protein VP2</a>.<br><br />
<br><br />
<br />
<br />
<br />
</p><br />
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<a name="arming"><h2>Arming</h2></a><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
</p><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
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<a name="superconstruct"><h2>All-in-One</h2></a><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
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Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion
Team:Freiburg Bioware/Project/Targeting Fusion
2010-10-27T14:01:34Z
<p>Hanna: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
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<p class="MsoToc2"><span lang="DE"><a<br />
href="#_Toc275953546"><span lang="EN-US">Tumor<br />
Targeting by N-terminal Fusion to Virus Capsid<br />
</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953547"><span lang="EN-US">Design</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953548"><span lang="EN-US">VP2<br />
Fusion</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953549"><span lang="EN-US">VP1<br />
Insertion</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953550"><span lang="EN-US">Cloning<br />
Verified by Colony PCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953551"><span lang="EN-US">Fluorescence<br />
Analysis of Viral Stocks</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953552"><span lang="EN-US">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953553"><span lang="EN-US">Nuclear<br />
Localization by Fluorescence Microscopy</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953554"><span lang="EN-US">Transduction<br />
Efficacy by Flow Cytometry</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953555"><span lang="EN-US">Infectious<br />
Titer by qPCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953556"><span lang="EN-US">Killing<br />
cells: Time-Lapse</span></a></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h2><a name="_Toc275953546">Tumor Targeting by<br />
N-terminal Fusion to Virus Capsid<br />
</a></h2><br />
<h3><a name="_Toc275953547">Design</a></h3><br />
<h4><a name="_Toc275953548">VP2 Fusion</a></h4><br />
<p class="MsoNormal">The Freiburg2010 team fused motifs<br />
which are desired to be<br />
surface exposed to the N-terminus of VP2/3 open reading frame, avoiding<br />
steric<br />
hindrance by connecting them with linker. We ensured in frame coupling<br />
by<br />
designing or using the referring parts in the Freiburg RFC25 standard,<br />
thus<br />
creating fusion constructs via NgoMIV and AgeI. For retargeting AAV2<br />
virus<br />
particles towards EGFR over expressing cancer cells we investigated the<br />
Z<sub>EGFR:1907</sub><br />
Affibody and the E01 DARPin – in combination with VP2/3_587-KO (HSPG<br />
knock<br />
down) – as surface exposed motifs. We also created so called<br />
“all-in-one” virus<br />
particles by fusing VP2/3_His-Tag or VP2/3_BAP via Middle Linker to the<br />
Affibody. Purification or imaging approaches could be conducted by<br />
His-Tag or<br />
CFP motifs, respectively. All constructs were cloned downstream of the<br />
CMV<br />
promoter (Fig.1).</p><br />
<p class="MsoNormal"><b>Table 1: List of VP2 fusion<br />
parts</b></p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"></p><br />
<p class="MsoNormal"></p><br />
<br><br />
<p class="MsoNormal">&nbsp;&nbsp; <img<br />
alt="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"<br />
src="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"></p><br />
<p class="MsoNormal"><b>Figure 1: Cloning scheme of<br />
VP2 fusion</b></p><br />
<p class="MsoNormal">First of all we fused the Affibody<br />
ZEGFR:1907 to the Short-,<br />
Middle-, Long- or SEG Linker and cloned the resulting constructs<br />
downstream of the<br />
CMV promoter. Afterwards either unmodified VP2/3 or HSPG affinity knock<br />
down<br />
VP2/3 were fused to the different linkers. Further on CFP and His-Tag<br />
were<br />
coupled to VP2/3 by the Middle Linker (Fig. 2).</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p style="text-align: center;" class="MsoNormal"><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"<br />
src="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style=""><span style="">&nbsp; </span></span><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
2:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis.<br />
Fusion of VP2/3 to CMV_Affibody_Linker or CMV_CFP_MiddleLinker<br />
constructs.</span></p><br />
<p class="MsoNormal">We conducted three fragment ligations<br />
with the DARPin E01,<br />
MiddleLinker_VP2/3 or MiddleLinker_VP2/3(587KO) and pSB1C3_CMV plasmid<br />
(Fig.<br />
3).</p><br />
<p style="text-align: center;" class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
3:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis. <b style="">A)</b><br />
Three fragment ligation of DARPin<br />
fused to N-terminus of VP2/3. <b style="">B)</b><br />
Test digestion verified successful cloning.<o:p></o:p></span></p><br />
<p class="MsoNormal">For 100 % replacement of wildtype VP2<br />
the fusion plasmid was<br />
co-transfected to the Rep/Cap plasmid which contained a start codon<br />
mutation of<br />
VP2. Wildtype VP2 knock-out was achieved by site-directed mutagenesis<br />
(Fig. 4).<br />
</p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251659264; left: 0px; margin-left: 295px; margin-top: 26px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 4:<br />
Knock out of VP2</span></b> in the Cap coding region of<br />
Rep/Cap plasmid:<br />
Knock-out of start codon via site-directed mutagenesis (note the C in<br />
the sequence).</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h4><a name="_Toc275953549">VP1 Insertion</a></h4><br />
<p class="MsoNormal">We inserted three diffent motifs into<br />
the VP1 open reading<br />
frame. They were fused, together with the unique uspstream region of<br />
VP1<br />
(VP1up) and a nuclear localization sequence (NLS), according to the<br />
RFC25<br />
standard to VP2/3 (Fig. 2). Again, all of these parts were driven by<br />
CMV<br />
promoter.</p><br />
<p class="MsoNormal"><b>Table 2: List of VP1<br />
insertion parts</b></p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"<br />
src="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"><br />
</div><br />
<p class="MsoNormal"></p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
src="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
height="656" width="875"></p><br />
<p style="font-weight: bold;" class="MsoNormal">Figure<br />
5: Cloning scheme of VP1<br />
insertion</p><br />
<p class="MsoNormal">First VP1up-NLS was fused to the<br />
surface exposed motif –<br />
Affibody, His-Tag or mVenus – and the resulting constructs were cloned<br />
downstream of the CMV promoter. This was followed by either fusing<br />
unmodified<br />
VP2/3 or HSPG affinity knock down VP2/3 (Fig. 6) to the different kinds<br />
of<br />
motifs.</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="width: 450px; height: 362px;"<br />
src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"<br />
alt="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
6:<br />
Agarose Gel Electrophoresis.</span></b><span<br />
style="font-size: 10pt;"> Fusion of VP2/3(587KO) to<br />
pCMV_VP1up_NLS_ZEGFR:1907,<br />
pCMV_VP1up_NLS_6xHis or pCMV_VP1up_NLS_mVenus<o:p></o:p></span></p><br />
<p class="MsoNormal">We designed site-directed mutagenesis<br />
primer for the VP1<br />
start codon modification in the Rep/Cap plasmid in order to ensure 100<br />
%<br />
replacement with modified VP1 proteins (Fig. 7).</p><br />
<p style="text-align: center;" class="MsoNormal">&nbsp;<img<br />
style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"></p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251660288; left: 0px; margin-left: 252px; margin-top: 5px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 7:<br />
Knock out of VP1</span></b> in Cap coding region of Rep/Cap<br />
plasmid: Knock-out<br />
of start codon via site-directed mutagenesis.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953550">Cloning Verified by<br />
Colony PCR</a></h3><br />
<p class="MsoNormal">Big amounts of samples necessitated<br />
fast and exact analysis<br />
of cloning. For this purpose colony PCR was conducted. We verified<br />
successful<br />
VP2/3 fusion by using primer that exclusively bound to a specific<br />
location in<br />
the VP2/3 sequence (Fig. 8). In the case of successful ligation a 880<br />
bp<br />
fragment was amplified via Taq polymerase.</p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"><br />
</div><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 8:</span></b><br />
Primer Cap 3500 for and VP 4200 rev. Colony PCR using these primer<br />
results in a<br />
880 bp fragment</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 9 demonstrates a colony PCR<br />
example: VP2 fusion<br />
constructs containing VP2/3(587) sequence and the so called “All in<br />
One”<br />
constructs were cloned into pSB1C3_CMV. Successful cloning could be<br />
verified<br />
(positive control: pAAV_RC; negative control: pSB1C3_lITR).</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
9: Colony<br />
PCR</span></b><span style="font-size: 10pt;"><br />
of VP2<br />
N-terminal fusion (Affibody_Linker, CFP_MiddleLinker,<br />
His-Tag_MiddleLinker:<br />
samples 1 - 6) and “All-in-One” constructs (samples 7 – 10).<o:p></o:p></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953551">Fluorescence Analysis of<br />
Viral Stocks</a></h3><br />
<p class="MsoNormal">We wanted to develop a protocol for<br />
harvesting as much<br />
viruses as possible. For this purpose we took advantage of viruses<br />
containing<br />
mVenus fused to VP1. In order to find out whether most viruses were<br />
located in<br />
the cell pellet, cell suspensions were centrifuged at 10.000 g or<br />
20.000 g.<br />
Afterwards mVenus fluorescence was measured at 526 nm. The resulting<br />
values were<br />
compared to cell culture supernatant, 20.000 g centrifuged supernatant<br />
and<br />
resuspended cell pellet (Fig. 10).</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in; width: 700px; height: 525px;"<br />
alt="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"<br />
src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"><br />
</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
10: Fluorescence signal of mVenus at 526 nm</span></b>,<br />
inserted into the virus<br />
capsid. Transduced cells were resuspended, pelleted or supernatant was<br />
taken in<br />
order to determine the location of most viruses. Control: DMEM.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953552">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</a></h3><br />
<p class="MsoNormal">For further analysis, viral stocks<br />
originating from cell<br />
culture supernatant needed to be concentrated. Therefore<br />
ultrafiltration with<br />
protein concentrators (Sartorius VivaSpin 20; 20.000Da MWCO) was<br />
conducted.<br />
Figure 11 shows fluorescence microscopy image of an aliquot from<br />
ultrafiltrated<br />
cell culture supernatant / cell lysate containing<br />
VP1up_NLS_mVenus_VP2/3<br />
viruses.</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"<br />
src="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
11: Fluorescence Microscopy</span></b>. A) Brightfield<br />
picture of an aliquot of<br />
concentrated VP1up_NLS_mVenus_VP2/3 containing virus stock. B) At 505<br />
nm<br />
excitation mVenus fluorescence of the virus particles could be<br />
detected. C)<br />
Merged image</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953553">Nuclear Localization by<br />
Fluorescence Microscopy</a></h3><br />
<p class="MsoNormal">AAV-293 cells were transfected with a<br />
50:50 ratio of the<br />
Rep/Cap(VP1KO) to the CMV_VP1_NLS_mVenus_VP2/3 plasmid. We packaged<br />
mCherry,<br />
driven by the CMV promoter, into the virus capsids and followed protein<br />
expression<br />
via fluorescence microscopy. 30 hours post transfection mCherry<br />
fluorescence<br />
was detectable in the whole cytosol of the successfully transfected<br />
cells,<br />
demonstrating that DNA located between the AAV2 ITRs is already<br />
transcribed in<br />
the producer cell line. In contrast to that mVenus fluorescence signal<br />
could be<br />
observed only in the nuclei (Fig. 12). This indicated that the nuclear<br />
localization sequence targets the single VP1_NLS_mVenus_VP2/3 proteins<br />
efficiently to the cell nucleus, where assembly and packaging of the<br />
virus<br />
particles takes place.&nbsp; </p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
src="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
height="656" width="875"></p><br />
<p class="MsoNormal" style="line-height: normal;">&nbsp;<br><br />
<b style=""><span style="font-size: 10pt;">Figure<br />
12:<br />
Fluorescence Microscopy. A) </span></b><span<br />
style="font-size: 10pt;">Brightfiled picture. <b style="">B)</b><br />
Excitation at 555 nm showed mCherry signal in the cytosol. <b<br />
style="">C)</b> Excitation at 505 nm revealed mVenus<br />
fluorescence in the nuclei, indicating functionality of the nuclear<br />
localization sequence inserted – together with mVenus – into VP1. <b<br />
style="">D)</b> Merged image.</span></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style="font-size: 10pt;"><o:p></o:p></span></p><br />
<h3><a name="_Toc275953554">Transduction Efficacy by<br />
Flow Cytometry</a></h3><br />
<p class="MsoNormal">For determination of transduction<br />
efficacy flow cytometry<br />
analysis was conducted. The Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
was fused with SEG-, middle- and long<br />
linker to the VP2/3 open reading frame (ORF) in order to investigate<br />
differences in infection efficacy due to different linker lengths.<br />
250.000<br />
AAV-293 cells were transfected with 1 µg total DNA. Different ratios of<br />
VP2<br />
fusion constructs in respect to the Rep/Cap plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and two different cell<br />
lines,<br />
HT1080 and A431, were transduced with 1 mL virus stock. By<br />
encapsulating mVenus<br />
coding sequence, the amount of transduced cells could be determined via<br />
flow<br />
cytometry.</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"<br />
src="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 13: Flow<br />
cytometry.</span></b><span style=""> Test of<br />
transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">Middlelinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p style="text-align: center;" class="MsoNormal"> <img<br />
alt="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 14: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with<br />
HT1080 and A431 cells by detecting mVenus expression from <b>Z<sub>EGFR:1907</sub>_SEG_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p class="MsoNormal"<br />
style="line-height: normal; text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"<br />
src="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 15: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">LongLinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span><b><span style="font-size: 12pt;"><o:p></o:p></span></b></p><br />
<span style=""><o:p></o:p></span><br />
<p class="MsoNormal">Additionally viruses containing a<br />
His-Tag or CFP, fused via<br />
Middle Linker to the VP2/3 ORF, were analyzed. Figure 16 overviews<br />
transduction<br />
efficacy of all constructs, transfected in a 10:90, 25:75 and 50:50<br />
ratio in<br />
respect to Rep/Cap plasmid.&nbsp;&nbsp; </p><br />
<p style="text-align: center;" class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"<br />
src="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"></p><br />
<p class="MsoNormal"></p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"><br><br />
</div><br />
<p class="MsoNormal"><b>Figure 16: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 and A431 cells, infected with virus<br />
particles<br />
consisting of different ratios of VP2 fusion constructs in respect to<br />
Rep/Cap<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Transduction of HT1080 cells revealed<br />
that all viral<br />
particles regardless of which motifs inserted into the capsids remained<br />
infectious with efficacies up to 85 %. In general the amount of mVenus<br />
positive<br />
cells decreased only slightly when harboring more modified VP2<br />
subunits. This<br />
indicates that larger peptides could be inserted into the AAV2 capsids<br />
without<br />
affecting virus assembly and packaging. A431 cells, which overexpress<br />
EGF<br />
receptor, were generally transduced with reduced efficacy. </p><br />
<p class="MsoNormal">Further on the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
or mVenus were inserted into the VP1<br />
ORF together with a nuclear localization signal. 250.000 AAV-293 cells<br />
were<br />
transfected with 1 µg total DNA and different ratios of VP1 insertion<br />
constructs in respect to the Rep/Cap(VP1KO) plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and HT1080 and A431<br />
cells were<br />
transduced. 48 hours later the number of transduced, or to be precise<br />
mVenus<br />
positive, cells was determined via flow cytometry (Fig. 17 a, b).</p><br />
<div style="text-align: center;"><img<br />
alt="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"><br />
</div><br />
<p class="MsoNormal"><b>Figure 17 a: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 cells, infected with virus particles<br />
containing different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO) plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"></p><br />
<p class="MsoNormal"><b>Figure 17 b: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive A431 cells, infected with virus particles<br />
containing<br />
different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO)<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Again results revealed that all virus<br />
particles remained<br />
infectious, regardless of whether inserting the Affibody or mVenus.<br />
This<br />
indicated that VP1 tolerated larger peptides inserted downstream of its<br />
unique<br />
N-terminal region and that this modification still allowed virus<br />
assembly and<br />
packaging.</p><br />
<h3><a name="_Toc275953555">Infectious Titer by qPCR</a><br />
</h3><br />
<p class="MsoNormal">We transfected 250.000 AAV-293 cells<br />
with 1 µg of total DNA<br />
composed of equal amounts of Rep/Cap, pHelper and vector plasmid. VP2<br />
fusion or<br />
VP1 insertion plasmids were co-transfected with two different ratios in<br />
respect<br />
to Rep/Cap(VP1KO) or Rep/Cap(VP2KO):&nbsp; 25&nbsp;% VP2 fusion<br />
or VP1 insertion<br />
proteins allow better assembly and packaging of the virus particles,<br />
compared<br />
to 50 % VP2 fusion or VP1 insertion proteins which increase the chance<br />
of&nbsp;<br />
integration into the capsids. </p><br />
<p class="MsoNormal">We created viruses containing the Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
Affibody fused<br />
to VP2 or inserted into VP1 and the DARPin E01 fused to VP2. These<br />
types of<br />
AAV2 particles were produced in two versions: With or without HSPG<br />
binding<br />
affinity knock down (587KO). </p><br />
<p class="MsoNormal">Viruses were harvested three days<br />
post transfection. The genomic<br />
titer was determined via qPCR by amplification of a specific sequence<br />
located<br />
in the CMV promoter of the vector plasmid (Table 3).</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Table 3:<br />
Quantitative Real-Time PCR.</span></b> Determination of<br />
genomic titer. Data<br />
were corrected for negative control value.</p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border: medium none ; width: 450.2pt; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"<br />
width="600"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Co-transfected<br />
Construct</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Ratio</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Genomic<br />
Titer /1ml</span></b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Corrected<br />
For Negative Control</span></b></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,20E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,39E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,17E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,44E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,63E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,67E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,37E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,38E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,52E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,50E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6,98E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,30E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,25E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,65E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">4,36E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,93E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,00E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,99E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,55E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,39E+08</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We investigated transduction of<br />
different cell lines. For<br />
this purpose 100.000 HT1080, HeLa or A431 cells were seeded and<br />
transduced with<br />
50 µL virus stock and harvested 24 hours later.&nbsp; Infectious<br />
titers were<br />
determined via qPCR and normalized to the genomic titers (Fig. 17-19).</p><br />
<p class="MsoNormal">Figure 18 shows infection efficacy of<br />
DARPin exposing<br />
viruses. Transduction of HT1080 cells was almost not affected as long<br />
as<br />
binding to HSPG was not knocked down. HeLa cells were also infected<br />
less<br />
efficient compared to the controls. However, A431 cells which<br />
overexpress EGFR<br />
were not infected by the controls. Transduction is rescued by<br />
integration of<br />
the DARPin into the virus capsid. By additionally knocking down the<br />
HSPG<br />
binding affinity these cells are transduced 10 times better, reaching<br />
wild type<br />
capsid HT1080 infection efficacy. These results indicated that specific<br />
re-targeting of AAV2 virus particles towards EGFR over expressing tumor<br />
cells<br />
was achieved by N-terminal fusion of targeting motifs to VP2.<br><br />
<br><br />
<br><br />
</p><br />
<div style="text-align: center;"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
height="656" width="875"></div><br />
<p class="MsoNormal"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 18:<br />
DARPin E01 VP2 Fusion.</span></b> Infectious titers were<br />
determined with or<br />
without HSPG knock down for HT1080, HeLa and A431 cells.<br />
Control:Rep/Cap<br />
plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We additionally obtained similar<br />
results for Affibody<br />
exposing viruses: A431 cell transduction could also be rescued by<br />
capsid<br />
integration of the Affibody. Again infection efficacy was increased by<br />
knocking<br />
down the natural tropism of the AAV2 viruses towards HSPG (Fig. 19).<br />
These data<br />
emphasized the functionality of the VP2 fusion constructs for<br />
specifically<br />
targeting tumor cells for therapeutic or imaging<br />
applications.&nbsp; </p><br />
<p class="MsoNormal"></p><br />
<div style="text-align: center;"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
height="656" width="875"><br><br />
</div><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
19:<br />
Affibody Z<sub>EGFR:1907</sub> VP2 Fusion.</span></b><br />
Infectious titers were<br />
determined with or without HSPG knock down for HT1080, HeLa and A431<br />
cells.<br />
Control:Rep/Cap plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 20 repesents the infectivity<br />
of viruses containing<br />
VP1 inserted Affibody molecules in respect to three different cell<br />
types<br />
(HT1080, HeLa, A431). The data clearly shows that virus capsids<br />
assembled and<br />
remained infectious. These AAV2 particles also featured specific<br />
binding<br />
properties to A431 cells, validating the functionality of&nbsp; the<br />
VP1 insertion<br />
strategy. The two outlier (HeLa VP1up_NLS_Affibody_VP2/3 25:75<br />
&amp; HT1080<br />
VP1up_NLS_Affibody_VP2/3(587KO) 50:50) should be left unconsidered. </p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
height="656" width="875"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 20:<br />
Affibody Z<sub>EGFR:1907</sub> VP1 Insertion.</span></b><br />
Infectious titers were<br />
determined for 25:75 and 50:50 transfection ratios with or without HSPG<br />
knock<br />
down for HT1080, HeLa and A431 cells. Control:Rep/Cap plasmid with and<br />
without<br />
HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953556">Killing cells: Time-Lapse</a></h3><br />
<p class="MsoNormal">HT1080 and A431 cells were transduced<br />
with so called<br />
“All-in-One” viruses containing the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
fused to VP2/3(587KO_His-Tag) and<br />
packaged with the guanylate kinase fused to the thymidine kinase coding<br />
sequence (GMK-TK). A time series of pictures was started directly after<br />
adding<br />
20 µM Ganciclovir (Fig.21).</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
a: Time-lapse</span></b>. HT1080 (control) cells transduced<br />
with GMK-TK<br />
packaged viruses and treated with 20 µM Ganciclovir A) 0 hours, B) 7<br />
hours, C)<br />
15 hours and D) 23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p style="text-align: center;" class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"<br />
src="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
b: Time-lapse</span></b>. A431 cells transduced with GMK-TK<br />
packaged viruses<br />
and treated with 20 µM Ganciclovir A) 0 hours, B) 7 hours, C) 15 hours<br />
and D)<br />
23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Virus and Ganciclovir treatment only<br />
slightly affect the<br />
morphology of HT1080 cells. After 23&nbsp;hours of incubation in<br />
20µM<br />
Ganciclovir the cells had almost the same appearance as at time point<br />
zero<br />
(Fig. 21 a). In comparison to that A431 epidermoid carcinoma cells were<br />
efficiently killed after transduction and Ganciclovir add-on: After 23<br />
hours<br />
nearly all cells were lysed (Fig. 21 b). These results clearly<br />
demonstrate that<br />
we were able to specifically target EGFR over-expressing tumor cells<br />
via capsid<br />
integrated Affibody and that these transduced cells were efficiently<br />
killed by<br />
expressing the GMK-TK which converted prodrug Ganciclovir into its<br />
cell-toxic monophosphate.<br />
</p><br />
<br />
<br />
</html></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion
Team:Freiburg Bioware/Project/Targeting Fusion
2010-10-27T13:48:53Z
<p>Hanna: New page: {{:Team:Freiburg_Bioware/Head}} {{:Team:Freiburg_Bioware/jquery}} {{:Team:Freiburg_Bioware/menu_home}} <html> <p class="MsoToc2"><span lang="DE"><a href="#_Toc275953546"><span lang="EN-U...</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<br />
<html><br />
<p class="MsoToc2"><span lang="DE"><a<br />
href="#_Toc275953546"><span lang="EN-US">Highlighting<br />
N-terminal Fusion:</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953547"><span lang="EN-US">Design</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953548"><span lang="EN-US">VP2<br />
Fusion</span></a></span></p><br />
<p class="MsoToc4"><span lang="DE"><a<br />
href="#_Toc275953549"><span lang="EN-US">VP1<br />
Insertion</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953550"><span lang="EN-US">Cloning<br />
Verified by Colony PCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953551"><span lang="EN-US">Fluorescence<br />
Analysis of Viral Stocks</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953552"><span lang="EN-US">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953553"><span lang="EN-US">Nuclear<br />
Localization by Fluorescence Microscopy</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953554"><span lang="EN-US">Transduction<br />
Efficacy by Flow Cytometry</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953555"><span lang="EN-US">Infectious<br />
Titer by qPCR</span></a></span></p><br />
<p class="MsoToc3"><span lang="DE"><a<br />
href="#_Toc275953556"><span lang="EN-US">Killing<br />
cells: Time-Lapse</span></a></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h2><a name="_Toc275953546">Highlighting N-terminal<br />
Fusion:</a></h2><br />
<h3><a name="_Toc275953547">Design</a></h3><br />
<h4><a name="_Toc275953548">VP2 Fusion</a></h4><br />
<p class="MsoNormal">The Freiburg2010 team fused motifs<br />
which are desired to be<br />
surface exposed to the N-terminus of VP2/3 open reading frame, avoiding<br />
steric<br />
hindrance by connecting them with linker. We ensured in frame coupling<br />
by<br />
designing or using the referring parts in the Freiburg RFC25 standard,<br />
thus<br />
creating fusion constructs via NgoMIV and AgeI. For retargeting AAV2<br />
virus<br />
particles towards EGFR over expressing cancer cells we investigated the<br />
Z<sub>EGFR:1907</sub><br />
Affibody and the E01 DARPin – in combination with VP2/3_587-KO (HSPG<br />
knock<br />
down) – as surface exposed motifs. We also created so called<br />
“all-in-one” virus<br />
particles by fusing VP2/3_His-Tag or VP2/3_BAP via Middle Linker to the<br />
Affibody. Purification or imaging approaches could be conducted by<br />
His-Tag or<br />
CFP motifs, respectively. All constructs were cloned downstream of the<br />
CMV<br />
promoter (Fig.1).</p><br />
<p class="MsoNormal"><b>Table 1: List of VP2 fusion<br />
parts</b></p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bf/Freiburg10_SchemaVP2Fusion.PNG"></p><br />
<p class="MsoNormal"></p><br />
<br><br />
<p class="MsoNormal">&nbsp;&nbsp; <img<br />
alt="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"<br />
src="https://static.igem.org/mediawiki/2010/2/25/Freiburg10_Overview_VP2Fusion.png"></p><br />
<p class="MsoNormal"><b>Figure 1: Cloning scheme of<br />
VP2 fusion</b></p><br />
<p class="MsoNormal">First of all we fused the Affibody<br />
ZEGFR:1907 to the Short-,<br />
Middle-, Long- or SEG Linker and cloned the resulting constructs<br />
downstream of the<br />
CMV promoter. Afterwards either unmodified VP2/3 or HSPG affinity knock<br />
down<br />
VP2/3 were fused to the different linkers. Further on CFP and His-Tag<br />
were<br />
coupled to VP2/3 by the Middle Linker (Fig. 2).</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"<br />
src="https://static.igem.org/mediawiki/2010/6/6a/Freiburg10_GelVP2Fusion2tog.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style=""><span style="">&nbsp; </span></span><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
2:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis.<br />
Fusion of VP2/3 to CMV_Affibody_Linker or CMV_CFP_MiddleLinker<br />
constructs.</span></p><br />
<p class="MsoNormal">We conducted three fragment ligations<br />
with the DARPin E01,<br />
MiddleLinker_VP2/3 or MiddleLinker_VP2/3(587KO) and pSB1C3_CMV plasmid<br />
(Fig.<br />
3).</p><br />
<p class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/2/23/Freiburg10_GelVP2Fusion2tog2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
3:</span></b><span style="font-size: 10pt;"><br />
Agarose Gel Electrophoresis. <b style="">A)</b><br />
Three fragment ligation of DARPin<br />
fused to N-terminus of VP2/3. <b style="">B)</b><br />
Test digestion verified successful cloning.<o:p></o:p></span></p><br />
<p class="MsoNormal">For 100 % replacement of wildtype VP2<br />
the fusion plasmid was<br />
co-transfected to the Rep/Cap plasmid which contained a start codon<br />
mutation of<br />
VP2. Wildtype VP2 knock-out was achieved by site-directed mutagenesis<br />
(Fig. 4).<br />
</p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251659264; left: 0px; margin-left: 295px; margin-top: 26px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ec/Freiburg10_VP2KO.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 4:<br />
Knock out of VP2</span></b> in the Cap coding region of<br />
Rep/Cap plasmid:<br />
Knock-out of start codon via site-directed mutagenesis (note the C in<br />
the sequence).</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h4><a name="_Toc275953549">VP1 Insertion</a></h4><br />
<p class="MsoNormal">We inserted three diffent motifs into<br />
the VP1 open reading<br />
frame. They were fused, together with the unique uspstream region of<br />
VP1<br />
(VP1up) and a nuclear localization sequence (NLS), according to the<br />
RFC25<br />
standard to VP2/3 (Fig. 2). Again, all of these parts were driven by<br />
CMV<br />
promoter.</p><br />
<p class="MsoNormal"><b>Table 2: List of VP1<br />
insertion parts</b></p><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"<br />
src="https://static.igem.org/mediawiki/2010/1/1b/Freiburg10_VP1InsertionSchema.PNG"><br />
<p class="MsoNormal"></p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
src="https://static.igem.org/mediawiki/2010/6/64/Freiburg10_Overview_VP1Insertion.png"<br />
height="656" width="875"></p><br />
<p style="font-weight: bold;" class="MsoNormal">Figure<br />
5: Cloning scheme of VP1<br />
insertion</p><br />
<p class="MsoNormal">First VP1up-NLS was fused to the<br />
surface exposed motif –<br />
Affibody, His-Tag or mVenus – and the resulting constructs were cloned<br />
downstream of the CMV promoter. This was followed by either fusing<br />
unmodified<br />
VP2/3 or HSPG affinity knock down VP2/3 (Fig. 6) to the different kinds<br />
of<br />
motifs.</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"<br />
src="https://static.igem.org/mediawiki/2010/9/96/Freiburg10_Gel_VP1ins.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
6:<br />
Agarose Gel Electrophoresis.</span></b><span<br />
style="font-size: 10pt;"> Fusion of VP2/3(587KO) to<br />
pCMV_VP1up_NLS_ZEGFR:1907,<br />
pCMV_VP1up_NLS_6xHis or pCMV_VP1up_NLS_mVenus<o:p></o:p></span></p><br />
<p class="MsoNormal">We designed site-directed mutagenesis<br />
primer for the VP1<br />
start codon modification in the Rep/Cap plasmid in order to ensure 100<br />
%<br />
replacement with modified VP1 proteins (Fig. 7).</p><br />
<p class="MsoNormal">&nbsp;<img<br />
style="cursor: -moz-zoom-out;"<br />
alt="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/93/Freiburg10_VP1KO.PNG"></p><br />
<p class="MsoNormal"><span<br />
style="position: absolute; z-index: 251660288; left: 0px; margin-left: 252px; margin-top: 5px; width: 42px; height: 99px;"></span></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 7:<br />
Knock out of VP1</span></b> in Cap coding region of Rep/Cap<br />
plasmid: Knock-out<br />
of start codon via site-directed mutagenesis.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953550">Cloning Verified by<br />
Colony PCR</a></h3><br />
<p class="MsoNormal">Big amounts of samples necessitated<br />
fast and exact analysis<br />
of cloning. For this purpose colony PCR was conducted. We verified<br />
successful<br />
VP2/3 fusion by using primer that exclusively bound to a specific<br />
location in<br />
the VP2/3 sequence (Fig. 8). In the case of successful ligation a 880<br />
bp<br />
fragment was amplified via Taq polymerase.</p><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/b/b4/Freiburg10_Primer_ColonyPCR.png"><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 8:</span></b><br />
Primer Cap 3500 for and VP 4200 rev. Colony PCR using these primer<br />
results in a<br />
880 bp fragment</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 9 demonstrates a colony PCR<br />
example: VP2 fusion<br />
constructs containing VP2/3(587) sequence and the so called “All in<br />
One”<br />
constructs were cloned into pSB1C3_CMV. Successful cloning could be<br />
verified<br />
(positive control: pAAV_RC; negative control: pSB1C3_lITR).</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"<br />
src="https://static.igem.org/mediawiki/2010/f/fb/Freiburg10_ColonyPCR.png"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="font-size: 10pt;">Figure<br />
9: Colony<br />
PCR</span></b><span style="font-size: 10pt;"><br />
of VP2<br />
N-terminal fusion (Affibody_Linker, CFP_MiddleLinker,<br />
His-Tag_MiddleLinker:<br />
samples 1 - 6) and “All-in-One” constructs (samples 7 – 10).<o:p></o:p></span></p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953551">Fluorescence Analysis of<br />
Viral Stocks</a></h3><br />
<p class="MsoNormal">We wanted to develop a protocol for<br />
harvesting as much<br />
viruses as possible. For this purpose we took advantage of viruses<br />
containing<br />
mVenus fused to VP1. In order to find out whether most viruses were<br />
located in<br />
the cell pellet, cell suspensions were centrifuged at 10.000 g or<br />
20.000 g.<br />
Afterwards mVenus fluorescence was measured at 526 nm. The resulting<br />
values were<br />
compared to cell culture supernatant, 20.000 g centrifuged supernatant<br />
and<br />
resuspended cell pellet (Fig. 10).</p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"<br />
src="https://static.igem.org/mediawiki/2010/1/1e/Freiburg10_test_supernatant_pellet.png"<br />
height="656" width="875"> </p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
10: Fluorescence signal of mVenus at 526 nm</span></b>,<br />
inserted into the virus<br />
capsid. Transduced cells were resuspended, pelleted or supernatant was<br />
taken in<br />
order to determine the location of most viruses. Control: DMEM.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953552">Concentrating<br />
VP1UP_NLS_mVenus_VP2/3 Viruses</a></h3><br />
<p class="MsoNormal">For further analysis, viral stocks<br />
originating from cell<br />
culture supernatant needed to be concentrated. Therefore<br />
ultrafiltration with<br />
protein concentrators (Sartorius VivaSpin 20; 20.000Da MWCO) was<br />
conducted.<br />
Figure 11 shows fluorescence microscopy image of an aliquot from<br />
ultrafiltrated<br />
cell culture supernatant / cell lysate containing<br />
VP1up_NLS_mVenus_VP2/3<br />
viruses.</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"<br />
src="https://static.igem.org/mediawiki/2010/d/d9/Freiburg10_mVenusDrop.PNG"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure<br />
11: Fluorescence Microscopy</span></b>. A) Brightfield<br />
picture of an aliquot of<br />
concentrated VP1up_NLS_mVenus_VP2/3 containing virus stock. B) At 505<br />
nm<br />
excitation mVenus fluorescence of the virus particles could be<br />
detected. C)<br />
Merged image</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953553">Nuclear Localization by<br />
Fluorescence Microscopy</a></h3><br />
<p class="MsoNormal">AAV-293 cells were transfected with a<br />
50:50 ratio of the<br />
Rep/Cap(VP1KO) to the CMV_VP1_NLS_mVenus_VP2/3 plasmid. We packaged<br />
mCherry,<br />
driven by the CMV promoter, into the virus capsids and followed protein<br />
expression<br />
via fluorescence microscopy. 30 hours post transfection mCherry<br />
fluorescence<br />
was detectable in the whole cytosol of the successfully transfected<br />
cells,<br />
demonstrating that DNA located between the AAV2 ITRs is already<br />
transcribed in<br />
the producer cell line. In contrast to that mVenus fluorescence signal<br />
could be<br />
observed only in the nuclei (Fig. 12). This indicated that the nuclear<br />
localization sequence targets the single VP1_NLS_mVenus_VP2/3 proteins<br />
efficiently to the cell nucleus, where assembly and packaging of the<br />
virus<br />
particles takes place.&nbsp; </p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
src="https://static.igem.org/mediawiki/2010/6/65/Freiburg10_NLS_mVenus_mCherry.png"<br />
height="656" width="875"></p><br />
<p class="MsoNormal" style="line-height: normal;">&nbsp;<br><br />
<b style=""><span style="font-size: 10pt;">Figure<br />
12:<br />
Fluorescence Microscopy. A) </span></b><span<br />
style="font-size: 10pt;">Brightfiled picture. <b style="">B)</b><br />
Excitation at 555 nm showed mCherry signal in the cytosol. <b<br />
style="">C)</b> Excitation at 505 nm revealed mVenus<br />
fluorescence in the nuclei, indicating functionality of the nuclear<br />
localization sequence inserted – together with mVenus – into VP1. <b<br />
style="">D)</b> Merged image.</span></p><br />
<p class="MsoNormal" style="line-height: normal;"><span<br />
style="font-size: 10pt;"><o:p></o:p></span></p><br />
<h3><a name="_Toc275953554">Transduction Efficacy by<br />
Flow Cytometry</a></h3><br />
<p class="MsoNormal">For determination of transduction<br />
efficacy flow cytometry<br />
analysis was conducted. The Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
was fused with SEG-, middle- and long<br />
linker to the VP2/3 open reading frame (ORF) in order to investigate<br />
differences in infection efficacy due to different linker lengths.<br />
250.000<br />
AAV-293 cells were transfected with 1 µg total DNA. Different ratios of<br />
VP2<br />
fusion constructs in respect to the Rep/Cap plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and two different cell<br />
lines,<br />
HT1080 and A431, were transduced with 1 mL virus stock. By<br />
encapsulating mVenus<br />
coding sequence, the amount of transduced cells could be determined via<br />
flow<br />
cytometry.</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"<br />
src="https://static.igem.org/mediawiki/2010/8/8e/Freiburg10_FACS1.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 13: Flow<br />
cytometry.</span></b><span style=""> Test of<br />
transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">Middlelinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p class="MsoNormal"> <img<br />
alt="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9f/Freiburg10_FACS2.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 14: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with<br />
HT1080 and A431 cells by detecting mVenus expression from <b>Z<sub>EGFR:1907</sub>_SEG_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non-transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span></p><br />
<p class="MsoNormal" style="line-height: normal;"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"<br />
src="https://static.igem.org/mediawiki/2010/7/77/Freiburg10_FACS3.PNG"></p><br />
<p class="MsoNormal" style="line-height: normal;"><b<br />
style=""><span style="">Figure 15: Flow<br />
cytometry.</span></b><span style=""><br />
Investigating transduction efficiency with HT1080<br />
and A431 cells by detecting mVenus expression from <b style="">Z<sub>EGFR:1907</sub>_<span<br />
style="">LongLinker</span>_VP2/3</b><br />
virus particles (Transfection ratio: 50:50 in respect to Rep/Cap<br />
plasmid). A) Gating<br />
non transduced cells (control); subcellular debris and cellular<br />
aggregates can<br />
be distinguished from single cells by size, estimated via forward<br />
scatter (FS<br />
Lin) and granularity, estimated via side scatter (SS Lin). B) <b>:</b><br />
Non<br />
transduced cells plotted against mVenus fluorescence (Analytical gate<br />
was set<br />
such that 1% or fewer of negative control cells fell within the<br />
positive region<br />
(R6). C) Gating transduced cells. D) Transduced cells plotted against<br />
mVenus<br />
fluorescence, R10 comprised transduced, mVenus expressing cells. E)<br />
Overlay of<br />
non transduced (red) and transduced (green) cells plotted against<br />
mVenus<br />
fluorescence.</span><b><span style="font-size: 12pt;"><o:p></o:p></span></b></p><br />
<span style=""><o:p></o:p></span><br />
<p class="MsoNormal">Additionally viruses containing a<br />
His-Tag or CFP, fused via<br />
Middle Linker to the VP2/3 ORF, were analyzed. Figure 16 overviews<br />
transduction<br />
efficacy of all constructs, transfected in a 10:90, 25:75 and 50:50<br />
ratio in<br />
respect to Rep/Cap plasmid.&nbsp;&nbsp; </p><br />
<p class="MsoNormal">&nbsp;<img<br />
alt="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"<br />
src="https://static.igem.org/mediawiki/2010/e/e2/Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png"></p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/1/15/Freiburg10_FACS_VP2_A431.png"><br><br />
<b>Figure 16: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 and A431 cells, infected with virus<br />
particles<br />
consisting of different ratios of VP2 fusion constructs in respect to<br />
Rep/Cap<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Transduction of HT1080 cells revealed<br />
that all viral<br />
particles regardless of which motifs inserted into the capsids remained<br />
infectious with efficacies up to 85 %. In general the amount of mVenus<br />
positive<br />
cells decreased only slightly when harboring more modified VP2<br />
subunits. This<br />
indicates that larger peptides could be inserted into the AAV2 capsids<br />
without<br />
affecting virus assembly and packaging. A431 cells, which overexpress<br />
EGF<br />
receptor, were generally transduced with reduced efficacy. </p><br />
<p class="MsoNormal">Further on the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
or mVenus were inserted into the VP1<br />
ORF together with a nuclear localization signal. 250.000 AAV-293 cells<br />
were<br />
transfected with 1 µg total DNA and different ratios of VP1 insertion<br />
constructs in respect to the Rep/Cap(VP1KO) plasmid were<br />
co-transfected. 72<br />
hours post transfection viruses were harvested and HT1080 and A431<br />
cells were<br />
transduced. 48 hours later the number of transduced, or to be precise<br />
mVenus<br />
positive, cells was determined via flow cytometry (Fig. 17 a, b).</p><br />
<img<br />
alt="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/2/22/Freiburg10_FACS_VP1Ins_HT1080.png"><br />
<p class="MsoNormal"><b>Figure 17 a: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive HT1080 cells, infected with virus particles<br />
containing different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO) plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"<br />
src="https://static.igem.org/mediawiki/2010/5/5b/Freiburg10_FACS_VP1Ins_A431.png"></p><br />
<p class="MsoNormal"><b>Figure 17 b: Flow cytometry<br />
analysis</b>. Transduced and<br />
therefore mVenus positive A431 cells, infected with virus particles<br />
containing<br />
different ratios of VP1 insertion constructs in respect to<br />
Rep/Cap(VP1KO)<br />
plasmid. </p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Again results revealed that all virus<br />
particles remained<br />
infectious, regardless of whether inserting the Affibody or mVenus.<br />
This<br />
indicated that VP1 tolerated larger peptides inserted downstream of its<br />
unique<br />
N-terminal region and that this modification still allowed virus<br />
assembly and<br />
packaging.</p><br />
<h3><a name="_Toc275953555">Infectious Titer by qPCR</a><br />
</h3><br />
<p class="MsoNormal">We transfected 250.000 AAV-293 cells<br />
with 1 µg of total DNA<br />
composed of equal amounts of Rep/Cap, pHelper and vector plasmid. VP2<br />
fusion or<br />
VP1 insertion plasmids were co-transfected with two different ratios in<br />
respect<br />
to Rep/Cap(VP1KO) or Rep/Cap(VP2KO):&nbsp; 25&nbsp;% VP2 fusion<br />
or VP1 insertion<br />
proteins allow better assembly and packaging of the virus particles,<br />
compared<br />
to 50 % VP2 fusion or VP1 insertion proteins which increase the chance<br />
of&nbsp;<br />
integration into the capsids. </p><br />
<p class="MsoNormal">We created viruses containing the Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
Affibody fused<br />
to VP2 or inserted into VP1 and the DARPin E01 fused to VP2. These<br />
types of<br />
AAV2 particles were produced in two versions: With or without HSPG<br />
binding<br />
affinity knock down (587KO). </p><br />
<p class="MsoNormal">Viruses were harvested three days<br />
post transfection. The genomic<br />
titer was determined via qPCR by amplification of a specific sequence<br />
located<br />
in the CMV promoter of the vector plasmid (Table 3).</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Table 3:<br />
Quantitative Real-Time PCR.</span></b> Determination of<br />
genomic titer. Data<br />
were corrected for negative control value.</p><br />
<div align="center"><br />
<table class="MsoNormalTable"<br />
style="border: medium none ; width: 450.2pt; border-collapse: collapse;"<br />
border="1" cellpadding="0" cellspacing="0"<br />
width="600"><br />
<tbody><br />
<tr><br />
<td<br />
style="border: 1pt solid windowtext; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Co-transfected<br />
Construct</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Ratio</span></b></p><br />
</td><br />
<td<br />
style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 2.85pt 5.4pt; background: rgb(234, 241, 221) none repeat scroll 0% 50%; -moz-background-clip: initial; -moz-background-origin: initial; -moz-background-inline-policy: initial; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Genomic<br />
Titer /1ml</span></b></p><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center; line-height: normal;"<br />
align="center"><b><span style="color: black;">Corrected<br />
For Negative Control</span></b></p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,20E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">2,39E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,17E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Affibody_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,44E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,63E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">CFP_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,67E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,37E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6xHis_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,38E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,52E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,50E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">6,98E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_Affibody_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,30E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,25E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">VP1up_NLS_6xHis_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,65E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">4,36E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,93E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">25:75</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,00E+09</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">DARPin_MiddleLinker_VP2/3(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">50:50</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">3,99E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">1,55E+08</p><br />
</td><br />
</tr><br />
<tr><br />
<td<br />
style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 2.85pt 5.4pt; width: 191.8pt;"<br />
width="256"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">Control: Rep/Cap(587KO)</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 110.2pt;"<br />
nowrap="nowrap" width="147"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">100%</p><br />
</td><br />
<td<br />
style="border-style: none solid solid none; border-color: -moz-use-text-color windowtext windowtext -moz-use-text-color; border-width: medium 1pt 1pt medium; padding: 2.85pt 5.4pt; width: 148.2pt;"<br />
nowrap="nowrap" width="198"><br />
<p class="MsoNormal"<br />
style="margin-bottom: 0.0001pt; text-align: center;"<br />
align="center">5,39E+08</p><br />
</td><br />
</tr><br />
</tbody><br />
</table><br />
</div><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We investigated transduction of<br />
different cell lines. For<br />
this purpose 100.000 HT1080, HeLa or A431 cells were seeded and<br />
transduced with<br />
50 µL virus stock and harvested 24 hours later.&nbsp; Infectious<br />
titers were<br />
determined via qPCR and normalized to the genomic titers (Fig. 17-19).</p><br />
<p class="MsoNormal">Figure 18 shows infection efficacy of<br />
DARPin exposing<br />
viruses. Transduction of HT1080 cells was almost not affected as long<br />
as<br />
binding to HSPG was not knocked down. HeLa cells were also infected<br />
less<br />
efficient compared to the controls. However, A431 cells which<br />
overexpress EGFR<br />
were not infected by the controls. Transduction is rescued by<br />
integration of<br />
the DARPin into the virus capsid. By additionally knocking down the<br />
HSPG<br />
binding affinity these cells are transduced 10 times better, reaching<br />
wild type<br />
capsid HT1080 infection efficacy. These results indicated that specific<br />
re-targeting of AAV2 virus particles towards EGFR over expressing tumor<br />
cells<br />
was achieved by N-terminal fusion of targeting motifs to VP2.<br><br />
<br><br />
<br><br />
<img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
src="https://static.igem.org/mediawiki/2010/9/9c/Freiburg10_qPCR_DARPin.PNG"<br />
height="656" width="875"><br />
</p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 18:<br />
DARPin E01 VP2 Fusion.</span></b> Infectious titers were<br />
determined with or<br />
without HSPG knock down for HT1080, HeLa and A431 cells.<br />
Control:Rep/Cap<br />
plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">We additionally obtained similar<br />
results for Affibody<br />
exposing viruses: A431 cell transduction could also be rescued by<br />
capsid<br />
integration of the Affibody. Again infection efficacy was increased by<br />
knocking<br />
down the natural tropism of the AAV2 viruses towards HSPG (Fig. 19).<br />
These data<br />
emphasized the functionality of the VP2 fusion constructs for<br />
specifically<br />
targeting tumor cells for therapeutic or imaging<br />
applications.&nbsp; </p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
src="https://static.igem.org/mediawiki/2010/e/ea/Freiburg10_qPCR_VP2Fus.PNG"<br />
height="656" width="875"><br><br />
<b><span style="font-size: 10pt; line-height: 200%;">Figure<br />
19:<br />
Affibody Z<sub>EGFR:1907</sub> VP2 Fusion.</span></b><br />
Infectious titers were<br />
determined with or without HSPG knock down for HT1080, HeLa and A431<br />
cells.<br />
Control:Rep/Cap plasmid with and without HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Figure 20 repesents the infectivity<br />
of viruses containing<br />
VP1 inserted Affibody molecules in respect to three different cell<br />
types<br />
(HT1080, HeLa, A431). The data clearly shows that virus capsids<br />
assembled and<br />
remained infectious. These AAV2 particles also featured specific<br />
binding<br />
properties to A431 cells, validating the functionality of&nbsp; the<br />
VP1 insertion<br />
strategy. The two outlier (HeLa VP1up_NLS_Affibody_VP2/3 25:75<br />
&amp; HT1080<br />
VP1up_NLS_Affibody_VP2/3(587KO) 50:50) should be left unconsidered. </p><br />
<p class="MsoNormal"><br><br />
</p><br />
<p class="MsoNormal"><img style="cursor: -moz-zoom-in;"<br />
alt="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
src="https://static.igem.org/mediawiki/2010/b/bd/Freiburg10_qPCR_VP1Insnew.PNG"<br />
height="656" width="875"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 100%;">Figure 20:<br />
Affibody Z<sub>EGFR:1907</sub> VP1 Insertion.</span></b><br />
Infectious titers were<br />
determined for 25:75 and 50:50 transfection ratios with or without HSPG<br />
knock<br />
down for HT1080, HeLa and A431 cells. Control:Rep/Cap plasmid with and<br />
without<br />
HSPG knock down.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<h3><a name="_Toc275953556">Killing cells: Time-Lapse</a></h3><br />
<p class="MsoNormal">HT1080 and A431 cells were transduced<br />
with so called<br />
“All-in-One” viruses containing the Affibody Z<sub><span<br />
style="font-size: 12pt; line-height: 200%;">EGFR:1907</span></sub><br />
fused to VP2/3(587KO_His-Tag) and<br />
packaged with the guanylate kinase fused to the thymidine kinase coding<br />
sequence (GMK-TK). A time series of pictures was started directly after<br />
adding<br />
20 µM Ganciclovir (Fig.21).</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"<br />
src="https://static.igem.org/mediawiki/2010/7/7f/Freiburg10_TimeLapseHT1080.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
a: Time-lapse</span></b>. HT1080 (control) cells transduced<br />
with GMK-TK<br />
packaged viruses and treated with 20 µM Ganciclovir A) 0 hours, B) 7<br />
hours, C)<br />
15 hours and D) 23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal"><img<br />
alt="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"<br />
src="https://static.igem.org/mediawiki/2010/3/3e/Freiburg10_TimeLapseA431.png"></p><br />
<p class="MsoNormal"><b><span<br />
style="font-size: 10pt; line-height: 200%;">Figure 21<br />
b: Time-lapse</span></b>. A431 cells transduced with GMK-TK<br />
packaged viruses<br />
and treated with 20 µM Ganciclovir A) 0 hours, B) 7 hours, C) 15 hours<br />
and D)<br />
23 hours post transduction.</p><br />
<p class="MsoNormal">&nbsp;</p><br />
<p class="MsoNormal">Virus and Ganciclovir treatment only<br />
slightly affect the<br />
morphology of HT1080 cells. After 23&nbsp;hours of incubation in<br />
20µM<br />
Ganciclovir the cells had almost the same appearance as at time point<br />
zero<br />
(Fig. 21 a). In comparison to that A431 epidermoid carcinoma cells were<br />
efficiently killed after transduction and Ganciclovir add-on: After 23<br />
hours<br />
nearly all cells were lysed (Fig. 21 b). These results clearly<br />
demonstrate that<br />
we were able to specifically target EGFR over-expressing tumor cells<br />
via capsid<br />
integrated Affibody and that these transduced cells were efficiently<br />
killed by<br />
expressing the GMK-TK which converted prodrug Ganciclovir into its<br />
cell-toxic monophosphate.<br />
</p><br />
</html></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Project/Results
Team:Freiburg Bioware/Project/Results
2010-10-27T13:46:47Z
<p>Hanna: </p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}{{:Team:Freiburg_Bioware/jquery}}{{:Team:Freiburg_Bioware/menu_home}}<br />
<html><br />
<h1>Results</h1><br />
<div class="box box_full"><br />
<table><br />
<tr><br />
<td><br />
<a name="highlights"><h2>Highlights</h2></a><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
</p><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="box box_full"><br />
<table><br />
<tr><br />
<td><br />
<a name="modularization"><h2>Modularization</h2></a><br />
<p><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Modularization_Vector_Plasmid">Mod Vector Plasmid</a><br />
Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
</p><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="box box_full"><br />
<table><br />
<tr><br />
<td><br />
<a name="targeting"><h2>Targeting</h2></a><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies n<br />
<br><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Fusion">Targeting by Fusion</a><br><br />
<a href="https://2010.igem.org/Team:Freiburg_Bioware/Project/Targeting_Loops">Targeting via Loops</a><br><br />
<br />
</p><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<div class="box box_full"><br />
<table><br />
<tr><br />
<td><br />
<a name="arming"><h2>Arming</h2></a><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
</p><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="box box_full"><br />
<table><br />
<tr><br />
<td><br />
<a name="superconstruct"><h2>All-in-One</h2></a><br />
<p>Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Aenean commodo ligula eget dolor. Aenean massa. Cum sociis natoque penatibus et magnis dis parturient montes, nascetur ridiculus mus. Donec quam felis, ultricies nec, pellentesque eu, pretium quis, sem. Nulla consequat massa quis enim. Donec pede justo, fringilla vel, aliquet nec, vulputate eget, arcu. In enim justo, rhoncus ut, imperdiet a, venenatis vitae, justo. Nullam dictum felis eu pede mollis pretium. Integer tincidunt. Cras dapibus. Vivamus elementum semper nisi. Aenean vulputate eleifend tellus. Aenean leo ligula, porttitor eu, consequat vitae, eleifend ac, enim. Aliquam lorem ante, dapibus in, viverra quis, feugiat a, tellus. Phasellus viverra nulla ut metus varius laoreet. Quisque rutrum. Aenean imperdiet. Etiam ultricies nisi vel augue. Curabitur ullamcorper ultricies nisi. Nam eget dui. Etiam rhoncus. Maecenas tempus, tellus eget condimentum rhoncus, sem quam semper libero, sit amet adipiscing sem neque sed ipsum. Nam quam nunc, blandit vel, luctus pulvinar, hendrerit id, lorem. Maecenas nec odio et ante tincidunt tempus. Donec vitae sapien ut libero venenatis faucibus. Nullam quis ante. Etiam sit amet orci eget eros faucibus tincidunt. Duis leo. Sed fringilla mauris sit amet nibh. Donec sodales sagittis magna. Sed consequat, leo eget bibendum sodales, augue velit cursus nunc.<br />
</p><br />
</td><br />
<td><br />
<img src="https://static.igem.org/mediawiki/2010/b/ba/Freiburg10_Banner_Snippet_3.png" alt="banner"/><br />
</td><br />
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{{:Team:Freiburg_Bioware/Footer}}</div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T13:30:22Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
<h2>Time lapse</h2><br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Structure Modeling</h2><br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_600px.png]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_400px.png]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_scheme_400px.png]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]] **neues Bild hochladen!**<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 mVenusDrop.PNG]]<br />
<br />
[[Media:Freiburg10 qPCR DARPin.PNG]]<br />
<br />
[[Media:Freiburg10 FACS1.PNG]]<br />
<br />
[[Media:Freiburg10 FACS2.PNG]]<br />
<br />
[[Media:Freiburg10 FACS3.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Insnew.PNG]]<br />
<br />
[[Media:Freiburg10 qPCR VP2Fus.PNG]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/File:Freiburg10_mVenusDrop.PNG
File:Freiburg10 mVenusDrop.PNG
2010-10-27T13:29:41Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T13:26:05Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
<h2>Time lapse</h2><br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Structure Modeling</h2><br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_600px.png]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_400px.png]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_scheme_400px.png]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]] **neues Bild hochladen!**<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 qPCR DARPin.PNG]]<br />
<br />
[[Media:Freiburg10 FACS1.PNG]]<br />
<br />
[[Media:Freiburg10 FACS2.PNG]]<br />
<br />
[[Media:Freiburg10 FACS3.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Insnew.PNG]]<br />
<br />
[[Media:Freiburg10 qPCR VP2Fus.PNG]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/File:Freiburg10_qPCR_DARPin.PNG
File:Freiburg10 qPCR DARPin.PNG
2010-10-27T13:25:13Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_FACS2.PNG
File:Freiburg10 FACS2.PNG
2010-10-27T13:15:49Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T13:15:37Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
<h2>Time lapse</h2><br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Structure Modeling</h2><br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_600px.png]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_400px.png]]<br />
<br />
[[Media:Freiburg10_AAV_assembly_scheme_400px.png]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]] **neues Bild hochladen!**<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 FACS1.PNG]]<br />
<br />
[[Media:Freiburg10 FACS2.PNG]]<br />
<br />
[[Media:Freiburg10 FACS3.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Insnew.PNG]]<br />
<br />
[[Media:Freiburg10 qPCR VP2Fus.PNG]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/File:Freiburg10_FACS3.PNG
File:Freiburg10 FACS3.PNG
2010-10-27T13:13:17Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_FACS1.PNG
File:Freiburg10 FACS1.PNG
2010-10-27T13:12:45Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Project/Results/Targeting
Team:Freiburg Bioware/Project/Results/Targeting
2010-10-27T11:56:01Z
<p>Hanna: New page: {{:Team:Freiburg_Bioware/Head}} {{:Team:Freiburg_Bioware/jquery}} {{:Team:Freiburg_Bioware/menu_home}} <html> </html> {{:Team:Freiburg_Bioware/Footer}}</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<br />
<html><br />
<br />
<br />
<br />
</html><br />
<br />
{{:Team:Freiburg_Bioware/Footer}}</div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T02:52:35Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Image:Freiburg10_VirusProductionTimeLapsemVenus.gif | 600px]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]]<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Insnew.PNG]]<br />
<br />
[[Media:Freiburg10 qPCR VP2Fus.PNG]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/File:Freiburg10_qPCR_VP1Insnew.PNG
File:Freiburg10 qPCR VP1Insnew.PNG
2010-10-27T02:51:57Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T02:51:01Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Image:Freiburg10_VirusProductionTimeLapsemVenus.gif | 600px]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]]<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Ins.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP2Fus.PNG]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T02:50:24Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Image:Freiburg10_VirusProductionTimeLapsemVenus.gif | 600px]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]]<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Ins.png]]<br />
<br />
[[Media:Image:Freiburg10 qPCR VP2Fus.PNG]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/File:Freiburg10_qPCR_VP2Fus.PNG
File:Freiburg10 qPCR VP2Fus.PNG
2010-10-27T02:49:25Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_qPCR_VP1Ins.PNG
File:Freiburg10 qPCR VP1Ins.PNG
2010-10-27T02:49:03Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T02:41:53Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Image:Freiburg10_VirusProductionTimeLapsemVenus.gif | 600px]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]]<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
<br />
[[Media:Freiburg10 qPCR VP1Ins.png]]<br />
<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/Team:Freiburg_Bioware/Filelist
Team:Freiburg Bioware/Filelist
2010-10-27T02:40:47Z
<p>Hanna: /* Results */</p>
<hr />
<div>{{:Team:Freiburg_Bioware/Head}}<br />
{{:Team:Freiburg_Bioware/jquery}}<br />
{{:Team:Freiburg_Bioware/menu_home}}<br />
<html> <br />
<h1>File list<h1><br />
<br />
</html><br />
<br />
<h2>Modeling</h2><br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection02.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection03.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10 VirusProductionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionData01.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Image:Freiburg10_VirusProductionTimeLapsemVenus.gif | 600px]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemVenus.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.gif]]<br />
<br />
[[Media:Freiburg10_VirusProductionTimeLapsemCherry.png]]<br />
<br />
[[Media:Freiburg10_VirusInfectionLogo.png]]<br />
<br />
[[Media:Freiburg10_VirusProductionLogo.png]]<br />
<br />
[[Media:Freiburg10_StructureModelingLogo.png]]<br />
<br />
[[Media:Freiburg10_production_Venus_mCherry_Brightfield.mov]]<br />
<br />
[[Media:Freiburg10_rot250trans.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-60.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.gif]]<br />
<br />
[[Media:Freiburg10_AAVassem500-100.mov]]<br />
<br />
[[Media:Freiburg10_TimeLapse_HT10180_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_A431_TK_GMK_400px.gif]]<br />
<br />
[[Media:Freiburg10_AAV_complete.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_singleVP3surface.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_VP3star.jpg]]<br />
<br />
[[Media:Freiburg10_AAV_slice.gif]]<br />
<br />
[[Media:Freiburg10_AAV_sliceSur.gif]]<br />
<br />
[[Media:Freiburg10_PleaseWait.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_390px.gif]]<br />
<br />
[[Media:Freiburg10_TimeLapse_AAV-293_mVenus+GOI_mCherry_400px.gif]]<br />
<br />
<br />
<h2>Modularaization of GOI - Results</h2><br />
<br />
[[Media:Freiburg10_Overview_BBa_Vectorplasmid.png]]<br />
<br />
[[Media:Freiburg10_Overview_Assembly_GOI.png]]<br />
<br />
[[Media:Freiburg10_TheoreticalCloning_ITR_CMV_beta.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Intermediate_GOI.png]]<br />
<br />
[[Media:Freiburg10_Cloning_Full_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_grey_1_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_Overlay_mVenus_2_GOI.png]]<br />
<br />
[[Media:Freiburg10_Microscopy_phasecontrast_2_GOI.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction01.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction02.png]]<br />
<br />
[[Media:Freiburg10_VirusProduction03.png]]<br />
<br />
[[Media:Freiburg10_VirusInfection01.png]]<br />
<br />
[[Media:Freiburg10_FACS_woHGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_withHGH.png]]<br />
<br />
[[Media:Freiburg10_Diagram_hGH.png]]<br />
<br />
[[Media:Freiburg10_FACS_betaglobin.png]]<br />
<br />
[[Media:Freiburg10_FACS_withbetaglobin.png]]<br />
<br />
[[Media:Freiburg10 VirusInfectionScheme01.png]]<br />
<br />
[[Media:Freiburg10_Diagram_betaglobin.png.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pSB1c3_mVenus.png]]<br />
<br />
[[Media:Freiburg10_FACS_FULL_pAAV_mVenus.png]]<br />
<br />
[[Media:Freiburg10_Diagram_FULL_GOI.png]]<br />
<br />
<br />
<br />
<h2> Introduction to Adeno-Associated Virus Serotype 2</h2><br />
<br />
[[Media:Freiburg10_genomic_organisation_wt.png]]<br />
<br />
[[Media:Freiburg10_organization_ITRs.png]]<br />
<br />
[[Media:Freiburg10_Overview_AAV-2_replication.png]]<br />
<br />
[[Media:Freiburg10_regulation_viral_promotors.png]]<br />
<br />
[[Media:Freiburg10_p5_promoter_wtAAV2.png]]<br />
<br />
[[Media:Freiburg10_p5_TATA_less.png]]<br />
<br />
[[Media:Freiburg10_DNA_Loop_P5_to_sp1.png]]<br />
<br />
[[Media:Freiburg10_Rep_proteins_organization.png]]<br />
<br />
[[Media:Freiburg10_Crystal_structure_SF3helicase.png]]<br />
<br />
[[Media:Freiburg10_PLA2-mechanism.PNG]]<br />
<br />
[[Media:Freiburg10_PLA2-recognition_site.jpg]]<br />
<br />
[[Media:Freiburg10_VP1-3_overview.jpg]]<br />
<br />
[[Media:Freiburg10_Rep40.jpg]]<br />
<br />
<h2> Trafficking </h2><br />
<br />
[[Media:Freiburg10_viral_trafficking.png]]<br />
<br />
[[Media:Freiburg10_cellular_surface_HSPG.png]]<br />
<br />
[[Media:Freiburg10_Clathrin_triskelions.png]]<br />
<br />
<h2> Helper genes </h2><br />
<br />
[[Media:Freiburg10_Helper_free_system.png]]<br />
<br />
[[Media:Freiburg10_pHelper.png]]<br />
<br />
<h2> Gene therapy </h2><br />
<br />
[[Media:Freiburg10_overview_GDEPT.png]]<br />
<br />
[[Media:Freiburg10_Bystander_effect.png]]<br />
<br />
<br />
<h2> Cytosine deaminase </h2><br />
<br />
[[Media:Freiburg10_Flourocytosine_pathway.png]]<br />
<br />
<h2> Loop insertions </h2><br />
<br />
[[Media:Freiburg10_3D_AC_of_453_587_loops.png]]<br />
<br />
[[Media:Freiburg10_cloning_viral_bricks.png]]<br />
<br />
<br />
<h2> Tagging </h2><br />
<br />
[[Media:Freiburg10_BAP.png]]<br />
<br />
[[Media:Freiburg10_His.png]]<br />
<br />
<br />
<h2> Targeting </h2><br />
<br />
[[Media:Freiburg10_RGD_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_motif.png]]<br />
<br />
[[Media:Freiburg10_Z34C_Z38_ZDomain_helix.png]]<br />
<br />
[[Media:Freiburg10_HSPG.png]]<br />
<br />
[[Media:Freiburg10_HSPG_chemical_structure.png]]<br />
<br />
<h2> N-terminal fusion </h2><br />
===Results===<br />
[[Media:Freiburg10 Cloning VP2Fusion2.PNG]]<br />
[[Media:Freiburg10 Cloning VP2FusionH.PNG]]<br />
[[Media:Freiburg10 CloningGel DARPin.png]]<br />
[[Media:Freiburg10 CMCSchema.png]]<br />
[[Media:Freiburg10 ColonyPCR.png]]<br />
[[Media:Freiburg10 FACS VP1Ins A431.png]]<br />
[[Media:Freiburg10 FACS VP1Ins HT1080.png]]<br />
[[Media:Freiburg10 FACS VP2 A431.png]]<br />
[[Media:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png]]<br />
[[Media:Freiburg10 Gel VP1ins.png]]<br />
[[Media:Freiburg10 GelVP2Fusion2tog.PNG]]<br />
[[Media:Freiburg10 GelVP2Fusion2tog2.PNG]]<br />
[[Media:Freiburg10 LinkerSchema.png]]<br />
[[Media:Freiburg10 NLS mVenus mCherry.png]]<br />
[[Media:Freiburg10 NLSSchema.png]]<br />
[[Media:Freiburg10 Overview VP1Insertion.png]]<br />
[[Media:Freiburg10 Overview VP2Fusion.png]]<br />
[[Media:Freiburg10 Primer ColonyPCR.png]]<br />
[[Media:Freiburg10 qPCR VP1Ins.png]]<br />
[[Media:Freiburg10 SchemaVP2Fusion.PNG]]<br />
[[Media:Freiburg10 TargetingMotifSchema.png]]<br />
[[Media:Freiburg10 test supernatant pellet.png]]<br />
[[Media:Freiburg10 TestDARPin.png]]<br />
[[Media:Freiburg10 TimeLapseA431.png]]<br />
[[Media:Freiburg10 TimeLapseHT1080.png]]<br />
[[Media:Freiburg10 VP1InsertionSchema.PNG]]<br />
[[Media:Freiburg10 VP1KO.PNG]]<br />
[[Media:Freiburg10 VP1upSchema.png]]<br />
[[Media:Freiburg10 VP2KO.PNG]]<br />
[[Media:Freiburg10 VP23Schema.png]]<br />
<br />
<br />
[[Media:Freiburg10_N_terminal_fusion_approach.png]]<br />
<br />
[[Media:Freiburg10_Cotransfected_plasmids.png]]<br />
<br />
<br />
<h2> VP1 insertion </h2><br />
<br />
[[Media:Freiburg10_pSB1C3_VP1up_NLS_Fig27.png]]<br />
<br />
[[Media:Freiburg10_VP1_insertion.png]]<br />
<br />
<br />
<h2> Targeting molecules </h2><br />
<br />
[[Media:Freiburg10_Nucleotide_sequence_ZEGFR.png]]<br />
<br />
[[Media:Freiburg10_Darpin.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<br />
<h2> Favorite parts </h2><br />
<br />
[[Media:Freiburg10_IMAC.png]]<br />
<br />
[[Media:Freiburg10_effect_GCV.png]]<br />
<br />
[[Media:Freiburg10_viral_brick_587_KO_His.png]]<br />
<br />
[[Media:Freiburg10_psB1C3_ZEGFR_Linker_VP23.png]]<br />
<br />
[[Media:Freiburg10_Darpin_internal_repeat_modules.png]]<br />
<br />
[[Media:Freiburg10_Darpin_conserved_motifs.png]]<br />
<br />
[[Media:Freiburg10_Darpin_capping_modules.png]]<br />
<br />
<h2> HSPG KO </h2><br />
<br />
[[Media:Freiburg10 HSPG binding motif.png]]<br />
<br />
[[Media:Freiburg10 heparansulfate.PNG]]</div>
Hanna
http://2010.igem.org/File:Freiburg10_VP23Schema.png
File:Freiburg10 VP23Schema.png
2010-10-27T02:33:14Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_VP2KO.PNG
File:Freiburg10 VP2KO.PNG
2010-10-27T02:32:57Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_VP1upSchema.png
File:Freiburg10 VP1upSchema.png
2010-10-27T02:32:43Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_VP1KO.PNG
File:Freiburg10 VP1KO.PNG
2010-10-27T02:32:24Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_VP1InsertionSchema.PNG
File:Freiburg10 VP1InsertionSchema.PNG
2010-10-27T02:32:05Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_TimeLapseHT1080.png
File:Freiburg10 TimeLapseHT1080.png
2010-10-27T02:31:45Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_TimeLapseA431.png
File:Freiburg10 TimeLapseA431.png
2010-10-27T02:31:28Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_TestDARPin.png
File:Freiburg10 TestDARPin.png
2010-10-27T02:31:12Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_test_supernatant_pellet.png
File:Freiburg10 test supernatant pellet.png
2010-10-27T02:30:52Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_TargetingMotifSchema.png
File:Freiburg10 TargetingMotifSchema.png
2010-10-27T02:30:37Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_SchemaVP2Fusion.PNG
File:Freiburg10 SchemaVP2Fusion.PNG
2010-10-27T02:30:17Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_qPCR_VP1Ins.png
File:Freiburg10 qPCR VP1Ins.png
2010-10-27T02:29:53Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_Primer_ColonyPCR.png
File:Freiburg10 Primer ColonyPCR.png
2010-10-27T02:29:34Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_Overview_VP2Fusion.png
File:Freiburg10 Overview VP2Fusion.png
2010-10-27T02:29:15Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_Overview_VP1Insertion.png
File:Freiburg10 Overview VP1Insertion.png
2010-10-27T02:28:54Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_NLSSchema.png
File:Freiburg10 NLSSchema.png
2010-10-27T02:28:36Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_NLS_mVenus_mCherry.png
File:Freiburg10 NLS mVenus mCherry.png
2010-10-27T02:28:17Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_LinkerSchema.png
File:Freiburg10 LinkerSchema.png
2010-10-27T02:28:00Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_GelVP2Fusion2tog2.PNG
File:Freiburg10 GelVP2Fusion2tog2.PNG
2010-10-27T02:27:44Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_GelVP2Fusion2tog.PNG
File:Freiburg10 GelVP2Fusion2tog.PNG
2010-10-27T02:27:27Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_Gel_VP1ins.png
File:Freiburg10 Gel VP1ins.png
2010-10-27T02:27:12Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_FACS_VP2Fusion_HT1080_Diagramm.png
File:Freiburg10 FACS VP2Fusion HT1080 Diagramm.png
2010-10-27T02:26:49Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_FACS_VP2_A431.png
File:Freiburg10 FACS VP2 A431.png
2010-10-27T02:26:35Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_FACS_VP1Ins_HT1080.png
File:Freiburg10 FACS VP1Ins HT1080.png
2010-10-27T02:26:20Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_FACS_VP1Ins_A431.png
File:Freiburg10 FACS VP1Ins A431.png
2010-10-27T02:26:04Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna
http://2010.igem.org/File:Freiburg10_ColonyPCR.png
File:Freiburg10 ColonyPCR.png
2010-10-27T02:25:47Z
<p>Hanna: </p>
<hr />
<div></div>
Hanna