Team:Freiburg Bioware/Project/Results/Modularization Vector Plasmid

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Contents

Introduction to Modularization of Vectorplasmid. 1

Recombinant and Modular Vectorplasmid Carrying GOI 2

Cloning and Combination Strategies for the Vectorplasmid. 3

Testing functionality of Assembled Vectorplasmid. 7

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression. 7

Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression. 8

Influence of hGH terminator BioBrick on GOI Expression. 8

Influence of Beta-globin intron Biobrick on GOI Expression. 11

Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression. 14

Conclusion. 16

References. 17

Introduction to Modularization of Vectorplasmid

Producing recombinant virus particles for therapeutical means is, besides specifically target cells, purification and quantification assays of AAV-2, one intention of the Virus Construction Kit provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular toolkit, the complex components of AAV-2 were extracted and redesigned to match the iGEM standard. Functional activity was tested in cell culture.

Differing from the wildtype AAV-2 genome, the Helper Free System provided by Stratagene comprises three plasmids and a specialized production cell line. AAV-293 cells derived from the HEK cell line express the stably integrated E1A and E1B helper proteins for efficient virus production. The plasmid containing the inverted terminal repeats (ITRs) is encapsidated into the preformed capsids after production of single-stranded DNA therefore also known as vectorplasmid (pGOI). Promoter, beta-globin intron and the hGH terminator signal are flanked by the ITRs and serve in the host cell for regulation of transgene expression. In addition to that, the plasmid coding for the Rep and Cap proteins (pRC) can be provided in trans leading to a layer of specificity due to the fact that the two genes are not packaged into the capsid since lacking of the ITRs impairs encapsidation. Another advantage of the Helper Free System can be attributed to cotransfection of another helper plasmid (pHelper), which provides the necessary proteins normally obtained by superinfection with helper viruses such as adenovirus or herpes simplex virus. These helper genes are required for full viral assembly by regulating gene expression of Rep and Cap proteins.

Recombinant and Modular Vectorplasmid Carrying GOI

The iGEM team Freiburg_Bioware 2010 provides a modular Virus Construction Kit for therapeutical applications, quantification assays and purification approaches depending on capsid modifications and the gene of interest flanked by the inverted terminal repeats (ITRs. In order to produce BioBrick-compatible standardized biological parts, we reengineered the plasmids and added new components for gene therapy approaches and analysis of biological activity of assembled BioBrick parts. Each element required for intact and functional plasmids comprising the ITRs, a promoter, a putative enhancer element and the hGH terminator was PCR amplified and fused together de novo. As shown in Figure 1, the vectorplasmid was assembled with the produced BioBricks consisting of the left and right ITR (BBa_K404100 and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin intron (BBa_K404107), the gene of interests (fluorescent proteins mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).

Figure 1: Overview of the theoretical sequence of each BioBrick provided within the Virus Construction Kit for an intact and fully functional rAAV genome. The plasmid in the lowest panel was used for tumor killing in combination with plasmids coding for modified capsid proteins. More detailed infomartion about these constructs can be found under ‘Arming: Killing the tumor’ and ‘N-terminal fusion for Targeting’.

Cloning and Combination Strategies for the Vectorplasmid

Organization of the recombinant viral DNA was modified ensuring several layers of specificity to our systems including a tumor-specific promoter and suicide genes encoding prodrug convertases. In order to modularize the rAAV sequence, each plasmid element (Figure 1) was PCR-amplified and cloned into the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team Freiburg_Bioware 2010 performed three site-directed mutagenesis in the gene of interest TK30 (BBa_K404109) and cytosine deaminase (BBa_K404112) for deletion of PstI and NgoMIV iGEM site (for further information see the results page of ‘Arming – Killing the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich regions forming T-shaped hairpins during replication, PCR amplification was not possible. Hence a cloning strategy was developed by the iGEM team Freiburg in order to provide BioBrick-compatible ITRs (see ‘Method Development of Cloning Strategy for ITRs’).

In Figure 2 the schematic overview of the modularization process can be seen which has been followed to conduct the assembly steps required for functional vectorplasmids.

Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png

Figure 3: Assembly procedure for fusion of BioBricks and composite parts to a fully assembled and functional plasmid coding for your gene of interest. This plasmid can be cotransfected with two helper plasmids providing protein for assembly and encapsidating of the rAAV genome (your gene of interest) into the capsids.

The iGEM team Freiburg_Bioware provides two examples demonstrating the assembly procedure for constructing vectorplasmids. The first representative example is the fusion of the BioBrick part beta-globin to the composite parts containing the 5´ elements of the plasmids, which are left ITR and CMV or phTERT promoter, respectively.

As shown in Figure 3 the theoretical cloning performed for assembling the BioBricks beta-globin intron and leftITR_CMV together can be observed.

Figure 4: Theoretical cloning of the composite part leftITR_CMV to the beta-globin intron BioBrick leading to the plasmid leftITR_CMV_beta-globin intron.

The plasmids were digested with both XbaI and PstI (beta-globin intron: BBa_K404107) or SpeI and PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the preparative gel in Figure 4, the expected bands could be detected under UV light and the extracted DNA could be successfully ligated. Each assembly step for producing BioBrick intermediates was conducted following the same strategy.

$Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png$

Figure 5: Assembly intermediate in fusion of the vectorplasmids containing different promoters. Fusion of the BioBrick part beta-globin (BBa_K404107) intron to the composite parts leftITR_pCMV and leftITR_phTERT, respectively, was performed following the BioBrick assembly strategy by digesting the insert with PstI and XbaI and the vectors with SpeI and PstI. The left lane shows the expected fragment at around 560 bp which corresponds to the beta-globin intron fragment, in contrast to the two lanes in the center and on the right which correspond to linearized plasmids after digesting with above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix; Insert, pSB1C3_beta-globin intron; Vector pCMV, pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.

Separated fragments were extracted using the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated with T4-ligase. After ligation has been carried out, E. coli XL-1B cells were transformed and incubated over night at 37°C. Picking clones from the transformation plate was performed the following day and DYT medium was inoculated incubating overnight. Plasmid DNA was isolated and test digestion revealed that cloning was successful obtaining the composite part leftITR_CMV_beta-globin intron (BBa_K404117).

Plasmid production incorporating all required elements for transgene expression and genome encapsidation into empty viral capsids was performed by fusing the downstream elements consisting of the hGH terminator and right ITR to the intermediate part providing the gene of interest and the promoter fused to the left ITR. Figure 5 demonstrates the assembly performed with pSB1C3_leftITR_phTERT_beta-globin intron_mVenus and pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after digestion on the left lane fits to the hGH-terminator_rightITR length. The isolated fragments were ligated and successful assembly was confirmed by test digestion obtaining the vectorplasmid pSB1C3_leftITR_phTERT_beta-globin intron_mVenus_hGH_rightITR (BBa_K404124).

$Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png$

Figure 6: Modularization of the assembled vectorplasmid containing the phTERT promoter and mVenus as gene of interest. Fusion of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part to the composite parts pSB1C3_hGH_rightITR was performed following the BioBrick assembly strategy by digesting the insert with XbaI and PstI and the vector with SpeI and PstI. The left lane corresponds to linearized plasmid after digesting with above mentioned iGEM restriction sites whereas the right lane reveals an intensive band at around 650 bp confirming the expected size of 657 bp of hGH_rITR. M, GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.

Since cloning does not confirm biological activity, we analyzed the plasmids and their functional components, hGH terminator and beta-globin intron, in cell culture. Assembled plasmids have been cotransfected, using AAV-293 cells, which provide the stable integrated E1A and E1B genes, with helper plasmids required for capsid assembly and genome encapsidation (pRC and pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus particles containing the single stranded DNA were harvested 72-hours post transfection and HT1080 cells transduced with constant volumes of viral vectors. 48-hours post infection; transduced cells expressing the gene of interest were analyzed by flow cytometry. Facilitating and demonstrating the analysis of functionality of the assembled plasmid, mVenus was used in first place since fluorescent proteins enable facile visualization using fluorescent microscopy and flow cytometry analysis.

Testing functionality of Assembled Vectorplasmid

Fluorescence Microscopy of Target Cells Demonstrates GOI Expression

Qualitative analysis of mVenus expression by fluorescence microscopy was conducted using Axio Observer Z1 showing that transduced HT1080 cells and non-transduced cells could be easily distinguished. In Figure 6 cells were excited with 505nm and fluorescence emission at 536nm was detected. Therefore, successful infection of tumor cells by recombinant viral particles carrying the assembled vectorplasmid coding for mVenus could be demonstrated.

A

Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)

B

Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG

C

Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg

D

Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg

Figure 7: Fluorescence microscopy (Exciatation: 505nm, Emission: 536nm) was performed for detection of transduced cell expression mVenus. A:Cells detected in bright field picture B: Detection of mVenus expression can be observed.

Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression

Characterizing the function of the hGH terminator, the beta-globin intron and the complete plasmid, several approaches were conducted followed by analysis via flow cytometry.

Influence of hGH terminator BioBrick on GOI Expression

The iGEM team Freiburg provides the hGH plolyadenylation sequence within the ‘Virus Construction Kit’ due to the fact that almost every eukaryotic mRNA is processed at their 3´ and 5´end except for histone mRNAs (Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved sequences. First, the polyadenylation signal “AATAAA” which is recognized by the multiprotein complex and second the GT-rich region (downstream sequence element, DSE) which is located 30 nucleotides downstream of the cleavage site. The assembled 3´end-processing machinery cleaves the mRNA transcript immediately after a CA-nucleotide therefore defining the cleavage site (Danckwardt et al. 2008). Recombinant vectorplasmids were engineered containing the inverted terminal repeats (ITRs), a strong eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of interest with and without the hGH terminator signal. Transduction of HT1080 cells with constant volume of viral particles containing the vectorplasmids and measuring mVenus expression 24-hours post infection by flow cytometry demonstrated that transgene expression of the constructs lacking the hGH termination signal is significantly reduced as shown in Figure 7 and Figure 8 confirming the expected results that hGH is essential for mRNA processing. The iGEM team Freiburg_Bioware 2010 therefore suggests using the provided hGH termination signal within the Virus Construction Kit for optimal gene expression.

Vectorplasmid lacking hGH termination signal

Vectorplasmid containing hGH terminator signal

Figure 8: Flow cytometry analysis of vectorplasmids with and without hGH terminator. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells applied against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5). C: Gating transduced cells (R2 ≙R14) (used plasmids for transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR (BBa_K404127), pHelper, pRC). D: Transduced cells plotted against mVenus, R10 comprises transduced cells by detecting mVenus expression. E: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.F: Gating non-transduced cells (control) G: Non-transduced cells applied against mVenus. H: Gating transduced cells (R2 ≙R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.

Figure 9: Flow cytometry analysis of vectorplasmids with and without hGH terminator. YFP expression of viral genomes was determined by flow cytomery after 24-hour post infection. Results demonstrate that mVenus expression of vectorplasmids lacking the hGH terminator is reduced significantly proving that the polyadenylation signal is essential for viral gene expression using recombinant viral vectors engineered by using components of the Virus Construction Kit.

Influence of Beta-globin intron Biobrick on GOI Expression

Providing an element assumed to be an enhancer of transgene expression (Nott et al. 2003), the iGEM team Freiburg tested a beta-globin intron derived from the human beta globin gene which can be fused upstream of the desired gene of interest. The beta-globin intron BioBrick consists of a partial chimeric CMV promoter followed by the intron II of the beta-globin gene. The 3´end of the intron is fused to the first 25 bases of human beta globin gene exon 3. The beta globin intron BioBrick is assumed to enhance eukaryotic gene expression (Nott et al. 2003). Analysis was conducted as described for the hGH terminator experiment (see above). As shown in Figure 9 and Figure 10 the vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral genomes containing the beta-globin intron. Considering these results and taking into account that a constant volume of viral particles has been used for transduction, the difference between the construct containing and lacking the beta-globin intron is minimal. Since packaging efficiency of the AAV-2 decreases with increasing sizes of the insert (Dong et al. 1996), the iGEM team Freiburg_Bioware suggests using the beta-globin intron in dependence on the size of your transgene.

Vectorplasmid lacking beta-globin intron

Vectorplasmid containing beta-globin intron

Figure 10: Flow cytometry analysis of vectorplasmids with and without beta-globin intron. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells applied against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5). C: Gating transduced cells (R2 ≙R14) (used plasmids for transfection: GOI: pSB1C3_lITR_CMV_mVenus_hGH_rITR (BBa_K404128), pHelper, pRC). D: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression E: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus F: Gating non-transduced cells (control). G: Non-transduced cells applied against mVenus (R5).H: Gating transduced cells (R2 ≙R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells applied against mVenus.

Figure 11: Flow cytometry analysis of vectorplasmids with and without beta-globin intron. 48-hours post transfection, viral particles were harvested by freeze-thaw lysis and centrifugation followed by HT1080 transduction. YFP expression of vectorplasmids was determined by flow cytometry 24-hours post infection. The vectorplasmid missing the beta-globin intron showed a negligible difference in mVenus expression compared to viral plasmid containing the beta-globin intron.

Functionality of the Full Assembled Vectorplasmid Demonstrated by GOI Expression

After assembly of plasmids containing all required elements (see Figure 1), functionality was tested in cell culture. AAV-293 cells stably expressing E1A and E1B proteins were transfected with three plasmids (pHelper, pRC, pGOI). Virus particles were harvested 72-hours post-transfection and the tumor cell line HT1080 was transduced with the recombinant viral vectors encapsidating the gene of interest mVenus (BBa_I757008).

The iGEM team Freiburg_Bioware 2010 compared the standard-plasmid containing a subcloned mVenus (pAAV_mVenus, derived from the Stratagene system) with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by flow cytometry analysis are shown in Figure 11 and Figure 12. Comparing mVenus expression of the standard plasmid and the modified, assembled plasmid reveals that biological functionality of the reassembled plasmid was confirmed.

pSB1C3_mVenus (BBa_K404119)

pAAV_mVenus (Stratagene)

Figure 12: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. A: Gating non transduced cells (control); subcellular debris and clumps can be distinguished from single cells by size, estimated forward scatter (FS Lin) and granularity, estimated side scatter (SS Lin) B: Non transduced cells plotted against mVenus (Analytical gate was set such that 1% or fewer of negative control cells fell within the positive region (R5).C: Gating transduced cells (R2 ≙R14) (used plasmids for transfection: pGOI: pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus: BBa_K404119), pHelper, pRC. D: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. E: Overlay of non-transduced (red) and transduced (green). F: Gating non transduced cells (control). G: Non-transduced cells plotted against mVenus (R5). H: Gating transduced cells (R14 ≙R2) (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). I: Transduced cells plotted against mVenus, R10 comprised transduced cells, by detecting mVenus expression. J: Overlay of non-transduced (red) and transduced (green) cells plotted against mVenus expression.

Figure 13: Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to standard plasmid provided by Stratagene. Fluorescence of the standard plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct was measured. As demonstrated mVenus expression is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus construct.

Conclusion

Idea of the modular ‘Virus Construction Kit’ is to provide all required elements for producing recombinant, functional virus particles delivering encapsidated genes of interest to specific cells. First step was to modify and modularize the vectorplasmid comprising basically the cis-elements for replication (ITRs), a strong eukaryotic or tissue specific promoter (pCMV or phTERT), the gene of interest (fluorescent proteins or suicide genes) and the hGH termination signal. Each element was successfully cloned and reassembled resulting in functional vectorplasmids determined by flow cytometry and fluorescence microscopy analyses. Experiments have been performed with mVenus since measurement of fluorescent proteins can be easily performed and visualized. Considering the results, the iGEM team Freiburg_Bioware 2010 then tested the construct containing the suicide genes thymidine kinase and cytosine deaminase. Further details demonstrating efficient tumor killing, using prodrug-activating systems, see results page ‘Arming – Killing the tumor’.

References

Danckwardt, S., Hentze, M.W. & Kulozik, A.E., 2008. 3' end mRNA processing: molecular mechanisms and implications for health and disease. The EMBO journal, 27(3), 482-98. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18256699.

Dong, J.Y., Fan, P.D. & Frizzell, R.a., 1996. Quantitative analysis of the packaging capacity of recombinant adeno-associated virus. Human gene therapy, 7(17), 2101-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.

Millevoi, S. et al., 2006. An interaction between U2AF 65 and CF I(m) links the splicing and 3' end processing machineries. The EMBO journal, 25(20), 4854-64. Available at: http://www.ncbi.nlm.nih.gov/pubmed/17024186.

Nott, A., Meislin, S.H. & Moore, M.J., 2003. A quantitative analysis of intron effects on mammalian gene expression. RNA (New York, N.Y.), 9(5), 607-17. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.

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+lang="EN-US">Fluorescence Microscopy of Target Cells Demonstrates GOI
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-lang=EN-US>Analysis of Target Cells by Flow Cytometry demonstrates GOI Expression</span><span
+lang="EN-US">Influence of hGH terminator BioBrick on GOI Expression</span><span
-style='color:windowtext;display:none;text-decoration:none'>. </span><span
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+Expression</span><span
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-lang=EN-US>Functionality of the Full Assembled Vectorplasmid Demonstrated by
+lang="EN-US">Conclusion</span><span
-GOI Expression</span><span style='color:windowtext;display:none;text-decoration:
+style="color: windowtext; display: none; text-decoration: none;">. </span><span
-none'>. </span><span
+style="color: windowtext; display: none; text-decoration: none;">16</span></a></span></p>
-style='color:windowtext;display:none;text-decoration:none'>14</span></a></span></p>
+<p class="MsoToc3"><span class="MsoHyperlink"><a href="#_Toc275800690"><span
+lang="EN-US">References</span><span
-<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275800689"><span
+style="color: windowtext; display: none; text-decoration: none;">. </span><span
-lang=EN-US>Conclusion</span><span style='color:windowtext;display:none;
+style="color: windowtext; display: none; text-decoration: none;">17</span></a></span></p>
-text-decoration:none'>. </span><span
+<p class="MsoNormal"><a name="_Toc275797953"><b>&nbsp;</b></a></p>
-style='color:windowtext;display:none;text-decoration:none'>16</span></a></span></p>
+<h2 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800680">Introduction
+to Modularization of Vectorplasmid</a></h2>
-<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275800690"><span
+<p class="MsoNormal"><span lang="EN-US">Producing recombinant virus
-lang=EN-US>References</span><span style='color:windowtext;display:none;
+particles for
-text-decoration:none'>. </span><span
+therapeutical means is, besides specifically target cells, purification
-style='color:windowtext;display:none;text-decoration:none'>17</span></a></span></p>
+and
+quantification assays of AAV-2, one intention of the Virus Construction
-<p class=MsoNormal><a name="_Toc275797953"><b>&nbsp;</b></a></p>
+Kit
+provided by the iGEM team Freiburg_Bioware 2010. For obtaining a
-<h2 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800680">Introduction to Modularization of Vectorplasmid</span></a></h2>
+modular
+toolkit, the complex components of AAV-2 were extracted and redesigned
-<p class=MsoNormal><span lang=EN-US>Producing recombinant virus particles for
+to match
-therapeutical means is, besides specifically target cells, purification and
-quantification assays of AAV-2, one intention of the Virus Construction Kit
-provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular
-toolkit, the complex components of AAV-2 were extracted and redesigned to match
 the iGEM standard. Functional activity was tested in cell culture.</span></p>
+<p class="MsoNormal"><span lang="EN-US">Differing from the wildtype
-<p class=MsoNormal><span lang=EN-US>Differing from the wildtype AAV-2 genome,
+AAV-2 genome,
-the Helper Free System provided by Stratagene comprises three plasmids and a
+the Helper Free System provided by Stratagene comprises three plasmids
-specialized production cell line. AAV-293 cells derived from the HEK cell line
+and a
-express the stably integrated E1A and E1B helper proteins for efficient virus
+specialized production cell line. AAV-293 cells derived from the HEK
-production. The plasmid containing the inverted terminal repeats (ITRs) is
+cell line
-encapsidated into the preformed capsids after production of single-stranded DNA
+express the stably integrated E1A and E1B helper proteins for efficient
+virus
+production. The plasmid containing the inverted terminal repeats (ITRs)
+is
+encapsidated into the preformed capsids after production of
+single-stranded DNA
 therefore also known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i>
-intron and the hGH terminator signal are flanked by the ITRs and serve in the
+intron and the hGH terminator signal are flanked by the ITRs and serve
-host cell for regulation of transgene expression. In addition to that, the
+in the
-plasmid coding for the Rep and Cap proteins (pRC) can be provided <i>in trans</i>
+host cell for regulation of transgene expression. In addition to that,
-leading to a layer of specificity due to the fact that the two genes are not
+the
-packaged into the capsid since lacking of the ITRs impairs encapsidation. Another
+plasmid coding for the Rep and Cap proteins (pRC) can be provided <i>in
-advantage of the Helper Free System can be attributed to cotransfection of
+trans</i>
+leading to a layer of specificity due to the fact that the two genes
+are not
+packaged into the capsid since lacking of the ITRs impairs
+encapsidation. Another
+advantage of the Helper Free System can be attributed to cotransfection
+of
 another helper plasmid (pHelper), which provides the necessary proteins
-normally obtained by superinfection with helper viruses such as adenovirus or
+normally obtained by superinfection with helper viruses such as
-herpes simplex virus. These helper genes are required for full viral assembly
+adenovirus or
+herpes simplex virus. These helper genes are required for full viral
+assembly
 by regulating gene expression of Rep and Cap proteins.</span></p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800681"></a><a
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800681"></a><a
 name="_Toc275797954">Recombinant and Modular Vectorplasmid Carrying
 GOI</a></h3>
+<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
-<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware 2010 provides
+provides
-a modular Virus Construction Kit for therapeutical applications, quantification
+a modular Virus Construction Kit for therapeutical applications,
-assays and purification approaches depending on capsid modifications and the
+quantification
-gene of interest flanked by the inverted terminal repeats (ITRs. In order to
+assays and purification approaches depending on capsid modifications
-produce BioBrick-compatible standardized biological parts, we reengineered the
+and the
-plasmids and added new components for gene therapy approaches and analysis of
+gene of interest flanked by the inverted terminal repeats (ITRs. In
-biological activity of assembled BioBrick parts. Each element required for
+order to
-intact and functional plasmids comprising the ITRs, a promoter, a putative
+produce BioBrick-compatible standardized biological parts, we
-enhancer element and the hGH terminator was PCR amplified and fused together <i>de
+reengineered the
-novo</i>.</span><span lang=EN-US> As shown in </span><span
+plasmids and added new components for gene therapy approaches and
-lang=EN-US>Figure 1</span><span lang=EN-US>, the vectorplasmid was assembled
+analysis of
-with the produced BioBricks consisting of the left and right ITR (BBa_K404100
+biological activity of assembled BioBrick parts. Each element required
-and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the
+for
-beta-globin intron (BBa_K404107), the gene of interests (fluorescent proteins
+intact and functional plasmids comprising the ITRs, a promoter, a
-mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112,
+putative
-mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).</span></p>
+enhancer element and the hGH terminator was PCR amplified and fused
+together <i>de
-<div align=center>
+novo</i>.</span><span lang="EN-US"> As shown in </span><span
+lang="EN-US">Figure 1</span><span lang="EN-US">, the vectorplasmid was
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+assembled
- style='border-collapse:collapse;border:none'>
+with the produced BioBricks consisting of the left and right ITR
- <tr style='height:522.25pt'>
+(BBa_K404100
-  <td width=589 valign=top style='width:441.75pt;border:solid windowtext 1.0pt;
+and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT:
-  padding:0cm 5.4pt 0cm 5.4pt;height:522.25pt'>
+BBa_K404106)) , the
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+beta-globin intron (BBa_K404107), the gene of interests (fluorescent
-cm;line-height:normal;page-break-after:avoid'><img width=580 height=692
+proteins
-  id="Grafik 5" src="Freiburg10_Modularization_GOI-Dateien/image001.jpg"></p>
+mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30:
-  <p class=MsoCaption style='text-indent:0cm'><span lang=EN-US>Figure </span><span lang=EN-US>1</span><span lang=EN-US>: </span><span lang=EN-US style='font-weight:
+BBa_K404112,
-  normal'>Overview of the theoretical sequence of each BioBrick provided within
+mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator
-  the Virus Construction Kit for an intact and fully functional rAAV genome.
+(BBa_K404108).</span></p>
-  The plasmid in the lowest panel was used for tumor killing in combination with
+<div align="center">
-  plasmids coding for modified capsid proteins. More detailed infomartion about
+<table class="MsoTableGrid"
-  these constructs can be found under ‘Arming: Killing the tumor’ and
+style="border: medium none ; border-collapse: collapse;" border="1"
-  ‘N-terminal fusion for Targeting’.</span></p>
+cellpadding="0" cellspacing="0">
-  </td>
+<tbody>
- </tr>
+<tr style="height: 522.25pt;">
+<td
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 441.75pt; height: 522.25pt;"
+valign="top" width="589">
+<p class="MsoNormal"
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+id="Grafik 5" src="Freiburg10_Modularization_GOI-Dateien/image001.jpg"
+height="692" width="580"></p>
+<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
+</span><span lang="EN-US">1</span><span lang="EN-US">: </span><span
+style="font-weight: normal;" lang="EN-US">Overview of the theoretical
+sequence of each BioBrick provided within the Virus Construction Kit
+for an intact and fully functional rAAV genome. The plasmid in the
+lowest panel was used for tumor killing in combination with plasmids
+coding for modified capsid proteins. More detailed infomartion about
+these constructs can be found under ‘Arming: Killing the tumor’ and
+‘N-terminal fusion for Targeting’.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+<h3><a name="_Toc275800682"></a><a name="_Toc275797955"><span
+lang="EN-US">Cloning
-<h3><a name="_Toc275800682"></a><a name="_Toc275797955"><span lang=EN-US>Cloning
+and Combination Strategies for the Vectorplasmid</span></a><span
-and Combination Strategies for the Vectorplasmid</span></a><span lang=EN-US> </span></h3>
+lang="EN-US"> </span></h3>
+<p class="MsoNormal"><span lang="EN-US">Organization of the recombinant
-<p class=MsoNormal><span lang=EN-US>Organization of the recombinant viral DNA was
+viral DNA was
-modified ensuring several layers of specificity to our systems including a
+modified ensuring several layers of specificity to our systems
-tumor-specific promoter and suicide genes encoding prodrug convertases. In
+including a
-order to modularize the rAAV sequence, each plasmid element (</span><span lang=EN-US>Figure 1</span><span lang=EN-US>) was PCR-amplified and cloned into
+tumor-specific promoter and suicide genes encoding prodrug convertases.
-the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team Freiburg_Bioware
+In
-performed three site-directed mutagenesis in the gene of interest TK30 (BBa_K404109)
+order to modularize the rAAV sequence, each plasmid element (</span><span
-and cytosine deaminase (</span><span lang=EN-US style='font-size:9.0pt;
+lang="EN-US">Figure 1</span><span lang="EN-US">) was PCR-amplified and
-line-height:200%'>BBa_K404112</span><span lang=EN-US>) for deletion of PstI and
+cloned into
-NgoMIV iGEM site (for further information see the results page of ‘Arming –
+the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
-Killing the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich
+Freiburg_Bioware
-regions forming T-shaped hairpins during replication, PCR amplification was not
+performed three site-directed mutagenesis in the gene of interest
-possible. Hence a cloning strategy was developed by the iGEM team Freiburg in
+TK30 (BBa_K404109)
-order to provide BioBrick-compatible ITRs (see ‘Method Development of Cloning
+and cytosine deaminase (</span><span
+style="font-size: 9pt; line-height: 200%;" lang="EN-US">BBa_K404112</span><span
+lang="EN-US">) for deletion of PstI and
+NgoMIV iGEM site (for further information see the results page of
+‘Arming –
+Killing the tumor’). Since the inverted terminal repeats (ITRs) are
+GC-rich
+regions forming T-shaped hairpins during replication, PCR amplification
+was not
+possible. Hence a cloning strategy was developed by the iGEM team
+Freiburg in
+order to provide BioBrick-compatible ITRs (see ‘Method Development of
+Cloning
 Strategy for ITRs’).</span></p>
+<p class="MsoNormal"><span lang="EN-US">In </span><span lang="EN-US">Figure
-<p class=MsoNormal><span lang=EN-US>In </span><span
+</span><span lang="EN-US"> the schematic overview of the
-lang=EN-US>Figure 2</span><span lang=EN-US> the schematic overview of the
+modularization process can be seen which has been followed to conduct
-modularization process can be seen which has been followed to conduct the
+the
 assembly steps required for functional vectorplasmids.</span></p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none'>
+<tbody>
- <tr style='height:26.15pt'>
+<tr style="height: 26.15pt;">
-  <td width=454 valign=top style='width:340.15pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt;height:26.15pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+valign="top" width="454">
-cm;line-height:normal;page-break-after:avoid'><img width=439 height=272
+<p class="MsoNormal"
-  id="Grafik 2" src="Freiburg10_Modularization_GOI-Dateien/image002.gif"
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-  alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"></p>
+id="Grafik 2" src="Freiburg10_Modularization_GOI-Dateien/image002.gif"
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275783119"><span
+alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
-  lang=EN-US>Figure </span></a><span lang=EN-US>3</span><span lang=EN-US>: </span><span
+height="272" width="439"></p>
-  lang=EN-US style='font-weight:normal'>Assembly procedure for fusion of
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  BioBricks and composite parts to a fully assembled and functional plasmid
+name="_Ref275783119"><span lang="EN-US">Figure </span></a><span
-  coding for your gene of interest. This plasmid can be cotransfected with two
+lang="EN-US">3</span><span lang="EN-US">: </span><span
-  helper plasmids providing protein for assembly and encapsidating of the rAAV
+style="font-weight: normal;" lang="EN-US">Assembly procedure for
-  genome (your gene of interest) into the capsids.</span></p>
+fusion of BioBricks and composite parts to a fully assembled and
-  </td>
+functional plasmid coding for your gene of interest. This plasmid can
- </tr>
+be cotransfected with two helper plasmids providing protein for
+assembly and encapsidating of the rAAV genome (your gene of interest)
+into the capsids.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
+provides two
-<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware provides two
+examples demonstrating the assembly procedure for constructing
-examples demonstrating the assembly procedure for constructing vectorplasmids.
+vectorplasmids.
 The first representative example is the fusion of the BioBrick part <i>beta-globin</i>
-to the composite parts containing the 5´ elements of the plasmids, which are
+to the composite parts containing the 5´ elements of the plasmids,
+which are
 left ITR and CMV or phTERT promoter, respectively.</span></p>
+<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">As
-<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>As shown in </span><span lang=EN-US>Figure 3</span><span lang=EN-US> the theoretical cloning performed
+shown in </span><span lang="EN-US">Figure 3</span><span lang="EN-US">
-for assembling the BioBricks <i>beta-globin </i>intron and leftITR_CMV together
+the theoretical cloning performed
+for assembling the BioBricks <i>beta-globin </i>intron and
+leftITR_CMV together
 can be observed. </span></p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none'>
+<tbody>
- <tr style='height:23.95pt'>
+<tr style="height: 23.95pt;">
-  <td width=467 valign=top style='width:349.9pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt;height:23.95pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+valign="top" width="467">
-cm;line-height:normal;page-break-after:avoid'><img width=452 height=530
+<p class="MsoNormal"
-  id="Grafik 74" src="Freiburg10_Modularization_GOI-Dateien/image003.gif"></p>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275783160"><span
+id="Grafik 74" src="Freiburg10_Modularization_GOI-Dateien/image003.gif"
-  lang=EN-US>Figure </span></a><span lang=EN-US>4</span><span lang=EN-US>:</span><span
+height="530" width="452"></p>
-  lang=EN-US> </span><span lang=EN-US style='font-weight:normal'>Theoretical
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  cloning of the composite part leftITR_CMV to the <i>beta-globin</i> intron
+name="_Ref275783160"><span lang="EN-US">Figure </span></a><span
-  BioBrick leading to the plasmid leftITR_CMV_<i>beta-globin</i> intron.</span></p>
+lang="EN-US">4</span><span lang="EN-US">:</span><span lang="EN-US"> </span><span
-  </td>
+style="font-weight: normal;" lang="EN-US">Theoretical cloning of the
- </tr>
+composite part leftITR_CMV to the <i>beta-globin</i> intron BioBrick
+leading to the plasmid leftITR_CMV_<i>beta-globin</i> intron.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+<p class="MsoNormal"><span lang="EN-US">The plasmids were digested with
+both XbaI
-<p class=MsoNormal><span lang=EN-US>The plasmids were digested with both XbaI
+and PstI (beta-globin intron: </span><span
-and PstI (beta-globin intron: </span><span lang=EN-US style='font-size:9.0pt;
+style="font-size: 9pt; line-height: 200%; color: black;" lang="EN-US">BBa_K404107</span><span
-line-height:200%;color:black'>BBa_K404107</span><span lang=EN-US>) or SpeI and
+lang="EN-US">) or SpeI and
-PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the preparative
+PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the
-gel in </span><span lang=EN-US>Figure 4</span><span lang=EN-US>, the expected
+preparative
+gel in </span><span lang="EN-US">Figure 4</span><span lang="EN-US">,
+the expected
 bands could be detected under UV light and the extracted DNA could be
-successfully ligated. Each assembly step for producing BioBrick intermediates
+successfully ligated. Each assembly step for producing BioBrick
+intermediates
 was conducted following the same strategy.</span></p>
+<div align="center">
-<div align=center>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none'>
+<tbody>
- <tr style='height:129.95pt'>
+<tr style="height: 129.95pt;">
-  <td width=417 valign=top style='width:312.75pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt;height:129.95pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+valign="top" width="417">
-cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
+<p class="MsoNormal"
-  <p class=MsoNormal align=right style='margin-bottom:0cm;margin-bottom:.0001pt;
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-  text-align:right;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
+lang="EN-US">&nbsp;</span></p>
-  width=397 height=194 id="Grafik 77"
+<p class="MsoNormal"
-  src="Freiburg10_Modularization_GOI-Dateien/image004.gif"
+style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-  alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"></p>
+align="right"><img id="Grafik 77"
-  <p class=MsoCaption style='text-indent:0cm'><span lang=EN-US>Figure </span><span lang=EN-US>5</span><span lang=EN-US>: Assembly intermediate in fusion of the
+src="Freiburg10_Modularization_GOI-Dateien/image004.gif"
-  vectorplasmids containing different promoters. </span><span lang=EN-US
+alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\09.09_Cloning_leftITR_beta to pCMV and phTERT.png"
-  style='font-weight:normal'>Fusion of the BioBrick part <i>beta-globin</i> (</span><span
+height="194" width="397"></p>
-  lang=EN-US style='color:black'>BBa_K404107</span><span lang=EN-US
+<p class="MsoCaption" style="text-indent: 0cm;"><span lang="EN-US">Figure
-  style='font-weight:normal'>) intron to the composite parts leftITR_pCMV and
+</span><span lang="EN-US">5</span><span lang="EN-US">: Assembly
-  leftITR_phTERT, respectively, was performed following the BioBrick assembly
+intermediate in fusion of the vectorplasmids containing different
-  strategy by digesting the insert with PstI and XbaI and the vectors with SpeI
+promoters. </span><span style="font-weight: normal;" lang="EN-US">Fusion
-  and PstI. The left lane shows the expected fragment at around 560 bp which
+of the BioBrick part <i>beta-globin</i> (</span><span
-  corresponds to the <i>beta-globin</i> intron fragment, in contrast to the two
+style="color: black;" lang="EN-US">BBa_K404107</span><span
-  lanes in the center and on the right which correspond to linearized plasmids
+style="font-weight: normal;" lang="EN-US">) intron to the composite
-  after digesting with above mentioned iGEM restriction sites. M, GeneRuler DNA
+parts leftITR_pCMV and leftITR_phTERT, respectively, was performed
-  ladder mix; Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,
+following the BioBrick assembly strategy by digesting the insert with
-  pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p>
+PstI and XbaI and the vectors with SpeI and PstI. The left lane shows
-  </td>
+the expected fragment at around 560 bp which corresponds to the <i>beta-globin</i>
- </tr>
+intron fragment, in contrast to the two lanes in the center and on the
+right which correspond to linearized plasmids after digesting with
+above mentioned iGEM restriction sites. M, GeneRuler DNA ladder mix;
+Insert, pSB1C3_<i>beta-globin</i> intron; Vector pCMV,
+pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+<p class="MsoNormal"><span lang="EN-US">Separated fragments were
+extracted using
-<p class=MsoNormal><span lang=EN-US>Separated fragments were extracted using
+the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated
-the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated with
+with
-T4-ligase. After ligation has been carried out, <i>E. coli</i> XL-1B cells were
+T4-ligase. After ligation has been carried out, <i>E. coli</i> XL-1B
+cells were
 transformed and incubated over night at 37°C. Picking clones from the
 transformation plate was performed the following day and DYT medium was
-inoculated incubating overnight. Plasmid DNA was isolated and test digestion
+inoculated incubating overnight. Plasmid DNA was isolated and test
-revealed that cloning was successful obtaining the composite part leftITR_CMV_<i>beta-globin</i>
+digestion
+revealed that cloning was successful obtaining the composite part
+leftITR_CMV_<i>beta-globin</i>
 intron (BBa_K404117).</span></p>
+<p class="MsoNormal"><span lang="EN-US">Plasmid production
-<p class=MsoNormal><span lang=EN-US>Plasmid production incorporating all
+incorporating all
-required elements for transgene expression and genome encapsidation into empty
+required elements for transgene expression and genome encapsidation
-viral capsids was performed by fusing the downstream elements consisting of the
+into empty
-hGH terminator and right ITR to the intermediate part providing the gene of
+viral capsids was performed by fusing the downstream elements
+consisting of the
+hGH terminator and right ITR to the intermediate part providing the
+gene of
 interest and the promoter fused to the left ITR. </span><span
-lang=EN-US>Figure 5</span><span lang=EN-US> demonstrates the assembly performed
+lang="EN-US">Figure 5</span><span lang="EN-US"> demonstrates the
+assembly performed
 with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus and
-pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after digestion on the
+pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after
-left lane fits to the hGH-terminator_rightITR length. The isolated fragments
+digestion on the
-were ligated and successful assembly was confirmed by test digestion obtaining
+left lane fits to the hGH-terminator_rightITR length. The isolated
-the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus_hGH_rightITR
+fragments
-(</span><span lang=EN-US style='line-height:200%;color:black'>BBa_K404124</span><span
+were ligated and successful assembly was confirmed by test digestion
-lang=EN-US>). </span></p>
+obtaining
+the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i>
-<div align=center>
+intron_mVenus_hGH_rightITR
+(</span><span style="line-height: 200%; color: black;" lang="EN-US">BBa_K404124</span><span
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+lang="EN-US">). </span></p>
- style='border-collapse:collapse;border:none'>
+<div align="center">
- <tr style='height:136.9pt'>
+<table class="MsoTableGrid"
-  <td width=411 valign=top style='width:308.4pt;border:solid windowtext 1.0pt;
+style="border: medium none ; border-collapse: collapse;" border="1"
-  padding:0cm 5.4pt 0cm 5.4pt;height:136.9pt'>
+cellpadding="0" cellspacing="0">
-  <p class=MsoNormal align=right style='margin-bottom:0cm;margin-bottom:.0001pt;
+<tbody>
-  text-align:right;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
+<tr style="height: 136.9pt;">
-  width=397 height=184 id="Grafik 80"
+<td
-  src="Freiburg10_Modularization_GOI-Dateien/image005.gif"
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"
-  alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"></p>
+valign="top" width="411">
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784510"><span
+<p class="MsoNormal"
-  lang=EN-US>Figure </span></a><span lang=EN-US>6</span><span lang=EN-US
+style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-  style='font-weight:normal'>: </span><span lang=EN-US>Modularization of the
+align="right"><img id="Grafik 80"
-  assembled vectorplasmid containing the phTERT promoter and mVenus as gene of
+src="Freiburg10_Modularization_GOI-Dateien/image005.gif"
-  interest.</span><span lang=EN-US style='font-weight:normal'> Fusion of the
+alt="Beschreibung: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
-  composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part  to the
+height="184" width="397"></p>
-  composite parts pSB1C3_hGH_rightITR was performed following the BioBrick
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  assembly strategy by digesting the insert with XbaI and PstI and the vector
+name="_Ref275784510"><span lang="EN-US">Figure </span></a><span
-  with SpeI and PstI. The left lane corresponds to linearized plasmid after
+lang="EN-US">6</span><span style="font-weight: normal;" lang="EN-US">:
-  digesting with above mentioned iGEM restriction sites whereas the right lane
+</span><span lang="EN-US">Modularization of the assembled
-  reveals an intensive band at around 650 bp confirming the expected size of
+vectorplasmid containing the phTERT promoter and mVenus as gene of
-bp of hGH_rITR. M, GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
+interest.</span><span style="font-weight: normal;" lang="EN-US"> Fusion
-  intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p>
+of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part
-  </td>
+to the composite parts pSB1C3_hGH_rightITR was performed following the
- </tr>
+BioBrick assembly strategy by digesting the insert with XbaI and PstI
+and the vector with SpeI and PstI. The left lane corresponds to
+linearized plasmid after digesting with above mentioned iGEM
+restriction sites whereas the right lane reveals an intensive band at
+around 650 bp confirming the expected size of 657 bp of hGH_rITR. M,
+GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
+intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
 </div>
+<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+<p class="MsoNormal"><span lang="EN-US">Since cloning does not confirm
+biological
-<p class=MsoNormal><span lang=EN-US>Since cloning does not confirm biological
 activity, we analyzed the plasmids and their functional components, hGH
-terminator and <i>beta-globin</i> intron, in cell culture. Assembled plasmids have
+terminator and <i>beta-globin</i> intron, in cell culture. Assembled
-been cotransfected, using AAV-293 cells, which provide the stable integrated
+plasmids have
-E1A and E1B genes, with helper plasmids required for capsid assembly  and
+been cotransfected, using AAV-293 cells, which provide the stable
+integrated
+E1A and E1B genes, with helper plasmids required for capsid assembly
+and
 genome encapsidation (pRC and pHelper) in a molar ratio of 1:1:1
-(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA were
+(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA
-harvested 72-hours post transfection and HT1080 cells transduced with constant
+were
-volumes of viral vectors. 48-hours post infection; transduced cells expressing
+harvested 72-hours post transfection and HT1080 cells transduced with
+constant
+volumes of viral vectors. 48-hours post infection; transduced cells
+expressing
 the gene of interest were analyzed by flow cytometry. Facilitating and
-demonstrating the analysis of functionality of the assembled plasmid, mVenus
+demonstrating the analysis of functionality of the assembled plasmid,
-was used in first place since fluorescent proteins enable facile visualization
+mVenus
+was used in first place since fluorescent proteins enable facile
+visualization
 using fluorescent microscopy and flow cytometry analysis.</span></p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800683"></a><a
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800683"></a><a
+name="_Toc275797956"><span lang="EN-US"><span
-name="_Toc275797956"><span lang=EN-US><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
-</span></span><span lang=EN-US>Testing functionality of Assembled Vectorplasmid</span></a></h3>
+lang="EN-US">Testing functionality of Assembled Vectorplasmid</span></a></h3>
+<h4><a name="_Toc275800684"></a><a name="_Toc275797957"><span
-<h4><a name="_Toc275800684"></a><a name="_Toc275797957"><span lang=EN-US>Fluorescence
+lang="EN-US">Fluorescence
 Microscopy of Target Cells Demonstrates GOI Expression</span></a></h4>
+<p class="MsoNormal"><span lang="EN-US">Qualitative analysis of mVenus
-<p class=MsoNormal><span lang=EN-US>Qualitative analysis of mVenus expression
+expression
-by fluorescence microscopy was conducted using Axio Observer Z1 showing that
+by fluorescence microscopy was conducted using Axio Observer Z1 showing
-transduced HT1080 cells and non-transduced cells could be easily distinguished.
+that
-In </span><span lang=EN-US>Figure 6</span><span lang=EN-US> cells were excited
+transduced HT1080 cells and non-transduced cells could be easily
-with 505nm and fluorescence emission at 536nm was detected. Therefore, successful
+distinguished.
-infection of tumor cells by recombinant viral particles carrying the assembled vectorplasmid
+In </span><span lang="EN-US">Figure 6</span><span lang="EN-US"> cells
+were excited
+with 505nm and fluorescence emission at 536nm was detected. Therefore,
+successful
+infection of tumor cells by recombinant viral particles carrying the
+assembled vectorplasmid
 coding for mVenus could be demonstrated. </span></p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=334 valign=top style='width:250.7pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 250.7pt;"
-cm;line-height:normal'><span lang=EN-US>A</span></p>
+valign="top" width="334">
-  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
+<p class="MsoNormal"
-  text-align:center;text-indent:0cm;line-height:normal'><img width=264
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-  height=218 id="Grafik 18"
+lang="EN-US">A</span></p>
-  src="Freiburg10_Modularization_GOI-Dateien/image006.jpg"
+<p class="MsoNormal"
-  alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"></p>
+style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-  </td>
+align="center"><img id="Grafik 18"
-  <td width=307 valign=top style='width:230.4pt;border:solid windowtext 1.0pt;
+src="Freiburg10_Modularization_GOI-Dateien/image006.jpg"
-  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
+alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+height="218" width="264"></p>
-cm;line-height:normal'><span lang=EN-US>B</span></p>
+</td>
-  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
+<td
-  text-align:center;text-indent:0cm;line-height:normal'><img width=242
+style="border-style: solid solid solid none; border-color: windowtext windowtext windowtext -moz-use-text-color; border-width: 1pt 1pt 1pt medium; padding: 0cm 5.4pt; width: 230.4pt;"
-  height=220 id="Grafik 19"
+valign="top" width="307">
-  src="Freiburg10_Modularization_GOI-Dateien/image007.jpg"
+<p class="MsoNormal"
-  alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"></p>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-  </td>
+lang="EN-US">B</span></p>
- </tr>
+<p class="MsoNormal"
- <tr>
+style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-  <td width=334 valign=top style='width:250.7pt;border:solid windowtext 1.0pt;
+align="center"><img id="Grafik 19"
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
+src="Freiburg10_Modularization_GOI-Dateien/image007.jpg"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"
-cm;line-height:normal'><span lang=EN-US>C</span></p>
+height="220" width="242"></p>
-  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
+</td>
-  text-align:center;text-indent:0cm;line-height:normal'><img width=258
+</tr>
-  height=195 id="Grafik 16"
+<tr>
-  src="Freiburg10_Modularization_GOI-Dateien/image008.jpg"
+<td
-  alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"></p>
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 250.7pt;"
-  </td>
+valign="top" width="334">
-  <td width=307 valign=top style='width:230.4pt;border-top:none;border-left:
+<p class="MsoNormal"
-  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-  padding:0cm 5.4pt 0cm 5.4pt'>
+lang="EN-US">C</span></p>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+<p class="MsoNormal"
-cm;line-height:normal'><span lang=EN-US>D</span></p>
+style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
+align="center"><img id="Grafik 16"
-  text-align:center;text-indent:0cm;line-height:normal'><img width=257
+src="Freiburg10_Modularization_GOI-Dateien/image008.jpg"
-  height=194 id="Grafik 17"
+alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
-  src="Freiburg10_Modularization_GOI-Dateien/image009.jpg"
+height="195" width="258"></p>
-  alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"></p>
+</td>
-  </td>
+<td
- </tr>
+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: 0cm 5.4pt; width: 230.4pt;"
- <tr>
+valign="top" width="307">
-  <td width=641 colspan=2 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+<p class="MsoNormal"
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784524"><span
+lang="EN-US">D</span></p>
-  lang=EN-US>Figure </span></a><span lang=EN-US>7</span><span lang=EN-US>: </span><span
+<p class="MsoNormal"
-  lang=EN-US style='font-weight:normal'>Fluorescence microscopy (Exciatation:
+style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
-nm, Emission: 536nm) was performed for detection of transduced cell
+align="center"><img id="Grafik 17"
-  expression mVenus. A:Cells detected in bright field picture B: Detection of
+src="Freiburg10_Modularization_GOI-Dateien/image009.jpg"
-  mVenus expression can be observed.</span></p>
+alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
-  </td>
+height="194" width="257"></p>
- </tr>
+</td>
+</tr>
+<tr>
+<td colspan="2"
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
+valign="top" width="641">
+<p class="MsoCaption" style="text-indent: 0cm;"><a
+name="_Ref275784524"><span lang="EN-US">Figure </span></a><span
+lang="EN-US">7</span><span lang="EN-US">: </span><span
+style="font-weight: normal;" lang="EN-US">Fluorescence microscopy
+(Exciatation: 505nm, Emission: 536nm) was performed for detection of
+transduced cell expression mVenus. A:Cells detected in bright field
+picture B: Detection of mVenus expression can be observed.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+<h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span
+lang="EN-US">Analysis
-<h4><a name="_Toc275800685"></a><a name="_Toc275797958"><span lang=EN-US>Analysis
 of Target Cells by Flow Cytometry demonstrates GOI Expression</span></a></h4>
+<p class="MsoNormal"><span lang="EN-US">Characterizing the function of
-<p class=MsoNormal><span lang=EN-US>Characterizing the function of the hGH
+the hGH
-terminator, the <i>beta-globin</i> intron and the complete plasmid, several
+terminator, the <i>beta-globin</i> intron and the complete plasmid,
+several
 approaches were conducted followed by analysis via flow cytometry. </span></p>
+<h5><a name="_Toc275800686"></a><a name="_Toc275797959"><span
-<h5><a name="_Toc275800686"></a><a name="_Toc275797959"><span lang=EN-US>Influence
+lang="EN-US">Influence
 of hGH terminator BioBrick on GOI Expression</span></a></h5>
+<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg provides
-<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg provides the hGH
+the hGH
-plolyadenylation sequence within the ‘Virus Construction Kit’ due to the fact
+plolyadenylation sequence within the ‘Virus Construction Kit’ due to
-that almost every eukaryotic mRNA is processed at their 3´ and 5´end except for
+the fact
-histone mRNAs </span><span
+that almost every eukaryotic mRNA is processed at their 3´ and 5´end
-lang=EN-US>(Millevoi et al. 2006)</span><span lang=EN-US>. Pre-mRNAs contain
+except for
-two canonical conserved sequences. First, the polyadenylation signal “AATAAA”
+histone mRNAs </span><span lang="EN-US">(Millevoi et al. 2006)</span><span
-which is recognized by the multiprotein complex and second the GT-rich region
+lang="EN-US">. Pre-mRNAs contain
-(downstream sequence element, DSE) which is located 30 nucleotides downstream
+two canonical conserved sequences. First, the polyadenylation signal
-of the cleavage site. The assembled 3´end-processing machinery cleaves the mRNA
+“AATAAA”
-transcript immediately after a CA-nucleotide therefore defining the cleavage
+which is recognized by the multiprotein complex and second the GT-rich
-site </span><span
+region
-lang=EN-US>(Danckwardt et al. 2008)</span><span lang=EN-US style='font-size:
+(downstream sequence element, DSE) which is located 30 nucleotides
-.0pt;line-height:200%'>. </span><span lang=EN-US>Recombinant vectorplasmids
+downstream
-were engineered containing the inverted terminal repeats (ITRs), a strong
+of the cleavage site. The assembled 3´end-processing machinery cleaves
-eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of interest
+the mRNA
-with and without the hGH terminator signal. Transduction of HT1080 cells with constant
+transcript immediately after a CA-nucleotide therefore defining the
-volume of viral particles containing the vectorplasmids and measuring mVenus
+cleavage
+site </span><span lang="EN-US">(Danckwardt et al. 2008)</span><span
+style="font-size: 12pt; line-height: 200%;" lang="EN-US">. </span><span
+lang="EN-US">Recombinant vectorplasmids
+were engineered containing the inverted terminal repeats (ITRs), a
+strong
+eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of
+interest
+with and without the hGH terminator signal. Transduction of HT1080
+cells with constant
+volume of viral particles containing the vectorplasmids and measuring
+mVenus
 expression 24-hours post infection by flow cytometry demonstrated that
-transgene expression of the constructs lacking the hGH termination signal is
+transgene expression of the constructs lacking the hGH termination
-significantly reduced as shown in </span><span
+signal is
-lang=EN-US>Figure 7</span><span lang=EN-US> and </span><span
+significantly reduced as shown in </span><span lang="EN-US">Figure 7</span><span
-lang=EN-US>Figure 8</span><span lang=EN-US> confirming the expected results
+lang="EN-US"> and </span><span lang="EN-US">Figure 8</span><span
-that hGH is essential for mRNA processing. The iGEM team Freiburg_Bioware 2010
+lang="EN-US"> confirming the expected results
-therefore suggests using the provided hGH termination signal within the Virus
+that hGH is essential for mRNA processing. The iGEM team
+Freiburg_Bioware 2010
+therefore suggests using the provided hGH termination signal within the
+Virus
 Construction Kit for optimal gene expression.</span></p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
-cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid lacking hGH
+valign="top" width="641">
-  termination signal</span></b></p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-cm;line-height:normal'><img width=629 height=408 id="Grafik 30"
+lang="EN-US">Vectorplasmid lacking hGH termination signal</span></b></p>
-  src="Freiburg10_Modularization_GOI-Dateien/image010.gif"></p>
+<p class="MsoNormal"
-  </td>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
- </tr>
+id="Grafik 30" src="Freiburg10_Modularization_GOI-Dateien/image010.gif"
- <tr>
+height="408" width="629"></p>
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+</td>
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
+</tr>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+<tr>
-cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid containing hGH
+<td
-  terminator signal</span></b></p>
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+valign="top" width="641">
-cm;line-height:normal;page-break-after:avoid'><img width=635 height=410
+<p class="MsoNormal"
-  id="Grafik 2049" src="Freiburg10_Modularization_GOI-Dateien/image011.gif"></p>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-  </td>
+lang="EN-US">Vectorplasmid containing hGH terminator signal</span></b></p>
- </tr>
+<p class="MsoNormal"
- <tr>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+id="Grafik 2049"
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
+src="Freiburg10_Modularization_GOI-Dateien/image011.gif" height="410"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+width="635"></p>
-cm;line-height:normal'><a name="_Ref275784539"><b><span lang=EN-US>Figure </span></b></a><b><span lang=EN-US>8</span></b><b><span lang=EN-US>:</span></b><span lang=EN-US> <b>Flow
+</td>
-  cytometry analysis of vectorplasmids with and without hGH terminator.</b> A:
+</tr>
-  Gating non transduced cells (control); subcellular debris and clumps can be
+<tr>
-  distinguished from single cells by size, estimated forward scatter (FS Lin)
+<td
-  and granularity, estimated side scatter (SS Lin) B: Non transduced cells
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
-  applied against mVenus (Analytical gate was set such that 1% or fewer of
+valign="top" width="641">
-  negative control cells fell within the positive region (R5). C: Gating transduced
+<p class="MsoNormal"
-  cells (R2 </span><span lang=EN-US style='font-family:"Cambria Math","serif"'>&#8793;</span><span
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a
-  lang=EN-US>R14) (used plasmids for transfection: GOI:
+name="_Ref275784539"><b><span lang="EN-US">Figure </span></b></a><b><span
-  pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR (BBa_K404127), pHelper, pRC).
+lang="EN-US">8</span></b><b><span lang="EN-US">:</span></b><span
-  D: Transduced cells plotted against mVenus, R10 comprises transduced cells by
+lang="EN-US"> <b>Flow cytometry analysis of vectorplasmids with and
-  detecting mVenus expression. E: Overlay of non-transduced (red) and
+without hGH terminator.</b> A: Gating non transduced cells (control);
-  transduced (green) cells applied against mVenus.F: Gating non-transduced
+subcellular debris and clumps can be distinguished from single cells by
-  cells (control) G: Non-transduced cells applied against mVenus. H: Gating
+size, estimated forward scatter (FS Lin) and granularity, estimated
-  transduced cells (R2 </span><span lang=EN-US style='font-family:"Cambria Math","serif"'>&#8793;</span><span
+side scatter (SS Lin) B: Non transduced cells applied against mVenus
-  lang=EN-US>R14) (used plasmids for transfection: GOI: reassembled pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+(Analytical gate was set such that 1% or fewer of negative control
-  (BBa_K404119), pHelper, pRC). I: Transduced cells applied against mVenus, R10
+cells fell within the positive region (R5). C: Gating transduced cells
-  comprised transduced cells, by detecting mVenus expression. J: Overlay of
+(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
-  non-transduced (red) and transduced (green) cells applied against mVenus.</span></p>
+lang="EN-US">≙</span><span lang="EN-US">R14) (used plasmids for
-  </td>
+transfection: GOI: pSB1C3_lITR_CMV_beta-globin intron_mVenus_rITR
- </tr>
+(BBa_K404127), pHelper, pRC). D: Transduced cells plotted against
+mVenus, R10 comprises transduced cells by detecting mVenus expression.
+E: Overlay of non-transduced (red) and transduced (green) cells applied
+against mVenus.F: Gating non-transduced cells (control) G:
+Non-transduced cells applied against mVenus. H: Gating transduced cells
+(R2 </span><span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;"
+lang="EN-US">≙</span><span lang="EN-US">R14) (used plasmids for
+transfection: GOI: reassembled
+pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119), pHelper,
+pRC). I: Transduced cells applied against mVenus, R10 comprised
+transduced cells, by detecting mVenus expression. J: Overlay of
+non-transduced (red) and transduced (green) cells applied against
+mVenus.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+<table class="MsoTableGrid"
+style="border: medium none ; border-collapse: collapse;" border="1"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+cellpadding="0" cellspacing="0">
- style='border-collapse:collapse;border:none'>
+<tbody>
- <tr>
+<tr>
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+valign="top" width="641">
-cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-cm;line-height:normal;page-break-after:avoid'><span lang=EN-US>                          </span><img
+lang="EN-US">&nbsp;</span></p>
-  width=473 height=355 id="Diagramm 3"
+<p class="MsoNormal"
-  src="Freiburg10_Modularization_GOI-Dateien/image012.gif"></p>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><span
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784545"><span
+lang="EN-US"> </span><img id="Diagramm 3"
-  lang=EN-US>Figure </span></a><span lang=EN-US>9</span><span lang=EN-US>: Flow
+src="Freiburg10_Modularization_GOI-Dateien/image012.gif" height="355"
-  cytometry analysis of vectorplasmids with and without hGH terminator.</span><span
+width="473"></p>
-  lang=EN-US style='font-weight:normal'> YFP expression of viral genomes was
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  determined by flow cytomery after 24-hour post infection. Results demonstrate
+name="_Ref275784545"><span lang="EN-US">Figure </span></a><span
-  that mVenus expression of vectorplasmids lacking the hGH terminator is
+lang="EN-US">9</span><span lang="EN-US">: Flow cytometry analysis of
-  reduced significantly proving that the polyadenylation signal is essential
+vectorplasmids with and without hGH terminator.</span><span
-  for viral gene expression using recombinant viral vectors engineered by using
+style="font-weight: normal;" lang="EN-US"> YFP expression of viral
-  components of the Virus Construction Kit.</span></p>
+genomes was determined by flow cytomery after 24-hour post infection.
-  </td>
+Results demonstrate that mVenus expression of vectorplasmids lacking
- </tr>
+the hGH terminator is reduced significantly proving that the
+polyadenylation signal is essential for viral gene expression using
+recombinant viral vectors engineered by using components of the Virus
+Construction Kit.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoCaption"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoCaption><span lang=EN-US>&nbsp;</span></p>
+<h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span
+lang="EN-US">Influence
-<h5><a name="_Toc275800687"></a><a name="_Toc275797960"><span lang=EN-US>Influence
 of <i>Beta-globin</i> intron Biobrick on GOI Expression</span></a></h5>
+<p class="MsoNormal"><span lang="EN-US">Providing an element assumed to
-<p class=MsoNormal><span lang=EN-US>Providing an element assumed to be an
+be an
-enhancer of transgene expression </span><span
+enhancer of transgene expression </span><span lang="EN-US">(Nott et
-lang=EN-US>(Nott et al. 2003)</span><span lang=EN-US>, the iGEM team Freiburg tested
+al. 2003)</span><span lang="EN-US">, the iGEM team Freiburg tested
-a beta-globin intron derived from the human <i>beta globin</i> gene which can
+a beta-globin intron derived from the human <i>beta globin</i> gene
-be fused upstream of the desired gene of interest. The beta-globin intron
+which can
-BioBrick consists of a partial chimeric CMV promoter followed by the intron II
+be fused upstream of the desired gene of interest. The beta-globin
-of the <i>beta-globin</i> gene. The 3´end of the intron is fused to the first 25
+intron
-bases of human <i>beta globin</i> gene exon 3. The <i>beta globin</i> intron
+BioBrick consists of a partial chimeric CMV promoter followed by the
-BioBrick is assumed to enhance eukaryotic gene expression </span><span lang=EN-US>(Nott et al. 2003)</span><span lang=EN-US>. Analysis was conducted as
+intron II
-described for the hGH terminator experiment (see above). As shown in </span><span lang=EN-US>Figure 9</span><span lang=EN-US> and </span><span
+of the <i>beta-globin</i> gene. The 3´end of the intron is fused to
-lang=EN-US>Figure 10</span><span lang=EN-US> the vectorplasmid missing the <i>beta-globin</i>
+the first 25
-intron showed a negligible difference in mVenus expression compared to viral
+bases of human <i>beta globin</i> gene exon 3. The <i>beta globin</i>
-genomes containing the <i>beta-globin</i> intron. Considering these results and
+intron
-taking into account that a constant volume of viral particles has been used for
+BioBrick is assumed to enhance eukaryotic gene expression </span><span
-transduction, the difference between the construct containing and lacking the
+lang="EN-US">(Nott et al. 2003)</span><span lang="EN-US">. Analysis
+was conducted as
+described for the hGH terminator experiment (see above). As shown in </span><span
+lang="EN-US">Figure 9</span><span lang="EN-US"> and </span><span
+lang="EN-US">Figure 10</span><span lang="EN-US"> the vectorplasmid
+missing the <i>beta-globin</i>
+intron showed a negligible difference in mVenus expression compared to
+viral
+genomes containing the <i>beta-globin</i> intron. Considering these
+results and
+taking into account that a constant volume of viral particles has been
+used for
+transduction, the difference between the construct containing and
+lacking the
 beta-globin intron is minimal. Since packaging efficiency of the AAV-2
-decreases with increasing sizes of the insert </span><span
+decreases with increasing sizes of the insert </span><span lang="EN-US">(Dong
-lang=EN-US>(Dong et al. 1996)</span><span lang=EN-US>, the iGEM team
+et al. 1996)</span><span lang="EN-US">, the iGEM team
-Freiburg_Bioware suggests using the <i>beta-globin </i>intron in dependence on
+Freiburg_Bioware suggests using the <i>beta-globin </i>intron in
+dependence on
 the size of your transgene.</span></p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 width=654
+style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
- style='width:490.75pt;border-collapse:collapse;border:none'>
+border="1" cellpadding="0" cellspacing="0" width="654">
- <tr style='height:2.5pt'>
+<tbody>
-  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
+<tr style="height: 2.5pt;">
-  padding:0cm 5.4pt 0cm 5.4pt;height:2.5pt'>
+<td
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 2.5pt;"
-cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid lacking <i>beta-globin</i>
+valign="top" width="654">
-  intron</span></b></p>
+<p class="MsoNormal"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-cm;line-height:normal'><img width=640 height=412 id="Grafik 55"
+lang="EN-US">Vectorplasmid lacking <i>beta-globin</i> intron</span></b></p>
-  src="Freiburg10_Modularization_GOI-Dateien/image013.gif"></p>
+<p class="MsoNormal"
-  </td>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
- </tr>
+id="Grafik 55" src="Freiburg10_Modularization_GOI-Dateien/image013.gif"
- <tr style='height:106.5pt'>
+height="412" width="640"></p>
-  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
+</td>
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:106.5pt'>
+</tr>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+<tr style="height: 106.5pt;">
-cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid containing <i>beta-globin</i>
+<td
-  intron</span></b></p>
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+valign="top" width="654">
-cm;line-height:normal;page-break-after:avoid'><img width=635 height=410
+<p class="MsoNormal"
-  id="Grafik 63" src="Freiburg10_Modularization_GOI-Dateien/image014.gif"></p>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-  </td>
+lang="EN-US">Vectorplasmid containing <i>beta-globin</i> intron</span></b></p>
- </tr>
+<p class="MsoNormal"
- <tr style='height:106.5pt'>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
+id="Grafik 63" src="Freiburg10_Modularization_GOI-Dateien/image014.gif"
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:106.5pt'>
+height="410" width="635"></p>
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784803"><span
+</td>
-  lang=EN-US>Figure </span></a><span lang=EN-US>10</span><span lang=EN-US>: Flow
+</tr>
-  cytometry analysis of vectorplasmids with and without <i>beta-globin</i>
+<tr style="height: 106.5pt;">
-  intron.  A</span><span lang=EN-US style='font-weight:normal'>: Gating non
+<td
-  transduced cells (control); subcellular debris and clumps can be
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 490.75pt; height: 106.5pt;"
-  distinguished from single cells by size, estimated forward scatter (FS Lin)
+valign="top" width="654">
-  and granularity, estimated side scatter (SS Lin) </span><span lang=EN-US>B</span><span
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  lang=EN-US style='font-weight:normal'>: Non transduced cells applied against
+name="_Ref275784803"><span lang="EN-US">Figure </span></a><span
-  mVenus (Analytical gate was set such that 1% or fewer of negative control
+lang="EN-US">10</span><span lang="EN-US">: Flow cytometry analysis of
-  cells fell within the positive region (R5). </span><span lang=EN-US>C</span><span
+vectorplasmids with and without <i>beta-globin</i> intron. A</span><span
-  lang=EN-US style='font-weight:normal'>: Gating transduced cells (R2 </span><span
+style="font-weight: normal;" lang="EN-US">: Gating non transduced
-  lang=EN-US style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
+cells (control); subcellular debris and clumps can be distinguished
-  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
+from single cells by size, estimated forward scatter (FS Lin) and
-  GOI: </span><span lang=EN-US>pSB1C3_lITR_CMV_mVenus_hGH_rITR (BBa_K404128)</span><span
+granularity, estimated side scatter (SS Lin) </span><span lang="EN-US">B</span><span
-  lang=EN-US style='font-weight:normal'>, pHelper, pRC). </span><span
+style="font-weight: normal;" lang="EN-US">: Non transduced cells
-  lang=EN-US>D</span><span lang=EN-US style='font-weight:normal'>: Transduced
+applied against mVenus (Analytical gate was set such that 1% or fewer
-  cells plotted against mVenus, R10 comprised transduced cells, by detecting
+of negative control cells fell within the positive region (R5). </span><span
-  mVenus expression </span><span lang=EN-US>E</span><span lang=EN-US
+lang="EN-US">C</span><span style="font-weight: normal;" lang="EN-US">:
-  style='font-weight:normal'>: Overlay of non-transduced (red) and transduced
+Gating transduced cells (R2 </span><span
-  (green) cells applied against mVenus </span><span lang=EN-US>F</span><span
+style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
-  lang=EN-US style='font-weight:normal'>: Gating non-transduced cells (control).
+lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
-  </span><span lang=EN-US>G</span><span lang=EN-US style='font-weight:normal'>:
+(used plasmids for transfection: GOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_mVenus_hGH_rITR
-  Non-transduced cells applied against mVenus (R5).</span><span lang=EN-US>H</span><span
+(BBa_K404128)</span><span style="font-weight: normal;" lang="EN-US">,
-  lang=EN-US style='font-weight:normal'>: Gating transduced cells (R2 </span><span
+pHelper, pRC). </span><span lang="EN-US">D</span><span
-  lang=EN-US style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
+style="font-weight: normal;" lang="EN-US">: Transduced cells plotted
-  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
+against mVenus, R10 comprised transduced cells, by detecting mVenus
-  GOI: reassembled </span><span lang=EN-US>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+expression </span><span lang="EN-US">E</span><span
-  (BBa_K404119)</span><span lang=EN-US style='font-weight:normal'>, pHelper, pRC).
+style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
-  </span><span lang=EN-US>I</span><span lang=EN-US style='font-weight:normal'>:
+(red) and transduced (green) cells applied against mVenus </span><span
-  Transduced cells applied against mVenus, R10 comprised transduced cells, by
+lang="EN-US">F</span><span style="font-weight: normal;" lang="EN-US">:
-  detecting mVenus expression. </span><span lang=EN-US>J</span><span
+Gating non-transduced cells (control). </span><span lang="EN-US">G</span><span
-  lang=EN-US style='font-weight:normal'>: Overlay of non-transduced (red) and
+style="font-weight: normal;" lang="EN-US">: Non-transduced cells
-  transduced (green) cells applied against mVenus.</span></p>
+applied against mVenus (R5).</span><span lang="EN-US">H</span><span
-  </td>
+style="font-weight: normal;" lang="EN-US">: Gating transduced cells
- </tr>
+(R2 </span><span
+style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
+lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
+(used plasmids for transfection: GOI: reassembled </span><span
+lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (BBa_K404119)</span><span
+style="font-weight: normal;" lang="EN-US">, pHelper, pRC). </span><span
+lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">:
+Transduced cells applied against mVenus, R10 comprised transduced
+cells, by detecting mVenus expression. </span><span lang="EN-US">J</span><span
+style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+(red) and transduced (green) cells applied against mVenus.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+<table class="MsoTableGrid"
+style="border: medium none ; width: 490.75pt; border-collapse: collapse;"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 width=654
+border="1" cellpadding="0" cellspacing="0" width="654">
- style='width:490.75pt;border-collapse:collapse;border:none'>
+<tbody>
- <tr style='height:90.2pt'>
+<tr style="height: 90.2pt;">
-  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
+<td
-  padding:0cm 5.4pt 0cm 5.4pt;height:90.2pt'>
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 90.2pt;"
-  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
+valign="top" width="654">
-  text-align:center;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
+<p class="MsoNormal"
-  width=450 height=332 id="Diagramm 57"
+style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-  src="Freiburg10_Modularization_GOI-Dateien/image015.gif"></p>
+align="center"><img id="Diagramm 57"
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784805"><span
+src="Freiburg10_Modularization_GOI-Dateien/image015.gif" height="332"
-  lang=EN-US>Figure </span></a><span lang=EN-US>11</span><span lang=EN-US>:
+width="450"></p>
-  Flow cytometry analysis of vectorplasmids with and without <i>beta-globin</i>
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  intron.</span><span lang=EN-US style='font-weight:normal'> 48-hours post
+name="_Ref275784805"><span lang="EN-US">Figure </span></a><span
-  transfection, viral particles were harvested by freeze-thaw lysis and
+lang="EN-US">11</span><span lang="EN-US">: Flow cytometry analysis of
-  centrifugation followed by HT1080 transduction. YFP expression of vectorplasmids
+vectorplasmids with and without <i>beta-globin</i> intron.</span><span
-  was determined by flow cytometry 24-hours post infection. The vectorplasmid
+style="font-weight: normal;" lang="EN-US"> 48-hours post transfection,
-  missing the beta-globin intron showed a negligible difference in mVenus
+viral particles were harvested by freeze-thaw lysis and centrifugation
-  expression compared to viral plasmid containing the beta-globin intron.</span></p>
+followed by HT1080 transduction. YFP expression of vectorplasmids was
-  </td>
+determined by flow cytometry 24-hours post infection. The vectorplasmid
- </tr>
+missing the beta-globin intron showed a negligible difference in mVenus
+expression compared to viral plasmid containing the beta-globin intron.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+<h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span
+lang="EN-US">Functionality
-<h5><a name="_Toc275800688"></a><a name="_Toc275797961"><span lang=EN-US>Functionality
 of the Full Assembled Vectorplasmid Demonstrated by GOI Expression</span></a><span
-lang=EN-US> </span></h5>
+lang="EN-US"> </span></h5>
+<p class="MsoNormal"><span lang="EN-US">After assembly of plasmids
-<p class=MsoNormal><span lang=EN-US>After assembly of plasmids containing all
+containing all
-required elements (see </span><span lang=EN-US>Figure 1</span><span lang=EN-US>),
+required elements (see </span><span lang="EN-US">Figure 1</span><span
-functionality was tested in cell culture. AAV-293 cells stably expressing E1A
+lang="EN-US">),
-and E1B proteins were transfected with three plasmids  (pHelper, pRC, pGOI).
+functionality was tested in cell culture. AAV-293 cells stably
-Virus particles were harvested 72-hours post-transfection and the tumor cell
+expressing E1A
-line HT1080 was transduced with the recombinant viral vectors encapsidating the
+and E1B proteins were transfected with three plasmids (pHelper, pRC,
+pGOI).
+Virus particles were harvested 72-hours post-transfection and the tumor
+cell
+line HT1080 was transduced with the recombinant viral vectors
+encapsidating the
 gene of interest mVenus (BBa_I757008).</span></p>
+<p class="MsoNormal"><span lang="EN-US">The iGEM team Freiburg_Bioware
-<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware 2010
-compared the standard-plasmid containing a subcloned mVenus (pAAV_mVenus,
+compared the standard-plasmid containing a subcloned mVenus
-derived from the Stratagene system) with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+(pAAV_mVenus,
-(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by flow
+derived from the Stratagene system) with the assembled plasmid
-cytometry analysis are shown in </span><span lang=EN-US>Figure 11</span><span
+pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-lang=EN-US> and </span><span lang=EN-US>Figure 12</span><span lang=EN-US>.
+(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by
-Comparing mVenus expression of the standard plasmid and the modified, assembled
+flow
-plasmid reveals that biological functionality of the reassembled plasmid was
+cytometry analysis are shown in </span><span lang="EN-US">Figure 11</span><span
+lang="EN-US"> and </span><span lang="EN-US">Figure 12</span><span
+lang="EN-US">.
+Comparing mVenus expression of the standard plasmid and the modified,
+assembled
+plasmid reveals that biological functionality of the reassembled
+plasmid was
 confirmed. </span></p>
+<table class="MsoTableGrid"
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+style="border: medium none ; border-collapse: collapse;" border="1"
- style='border-collapse:collapse;border:none'>
+cellpadding="0" cellspacing="0">
- <tr>
+<tbody>
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+<tr>
-  padding:0cm 5.4pt 0cm 5.4pt'>
+<td
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
-cm;line-height:normal'><b>pSB1C3_mVenus (BBa_K404119)</b></p>
+valign="top" width="641">
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+<p class="MsoNormal"
-cm;line-height:normal'><img width=634 height=409 id="Grafik 2068"
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>pSB1C3_mVenus
-  src="Freiburg10_Modularization_GOI-Dateien/image016.gif"></p>
+(BBa_K404119)</b></p>
-  </td>
+<p class="MsoNormal"
- </tr>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
- <tr>
+id="Grafik 2068"
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+src="Freiburg10_Modularization_GOI-Dateien/image016.gif" height="409"
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
+width="634"></p>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+</td>
-cm;line-height:normal'><b><span lang=EN-US>pAAV_mVenus (Stratagene)</span></b></p>
+</tr>
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+<tr>
-cm;line-height:normal;page-break-after:avoid'><img width=630 height=407
+<td
-  id="Grafik 89" src="Freiburg10_Modularization_GOI-Dateien/image017.gif"></p>
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
-  </td>
+valign="top" width="641">
- </tr>
+<p class="MsoNormal"
- <tr>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+lang="EN-US">pAAV_mVenus (Stratagene)</span></b></p>
-  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
+<p class="MsoNormal"
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784576"><span
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
-  lang=EN-US>Figure </span></a><span lang=EN-US>12</span><span lang=EN-US>: Flow
+id="Grafik 89" src="Freiburg10_Modularization_GOI-Dateien/image017.gif"
-  cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to
+height="407" width="630"></p>
-  standard plasmid provided by Stratagene. A</span><span lang=EN-US
+</td>
-  style='font-weight:normal'>: Gating non transduced cells (control);
+</tr>
-  subcellular debris and clumps can be distinguished from single cells by size,
+<tr>
-  estimated forward scatter (FS Lin) and granularity, estimated side scatter
+<td
-  (SS Lin) B: Non transduced cells plotted against mVenus (Analytical gate was
+style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 481.1pt;"
-  set such that 1% or fewer of negative control cells fell within the positive
+valign="top" width="641">
-  region (R5).C: Gating transduced cells (R2 </span><span lang=EN-US
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
+name="_Ref275784576"><span lang="EN-US">Figure </span></a><span
-  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
+lang="EN-US">12</span><span lang="EN-US">: Flow cytometry analysis of
-  pGOI: </span><span lang=EN-US>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+reassembled vectorplasmid (BBa_K404119) compared to standard plasmid
-  (pSB1C3_mVenus: </span><span lang=EN-US>BBa_K404119</span><span lang=EN-US
+provided by Stratagene. A</span><span style="font-weight: normal;"
-  style='color:#00B050;font-weight:normal'>)</span><span lang=EN-US
+lang="EN-US">: Gating non transduced cells (control); subcellular
-  style='font-weight:normal'>, pHelper, pRC. </span><span lang=EN-US>D</span><span
+debris and clumps can be distinguished from single cells by size,
-  lang=EN-US style='font-weight:normal'>: Transduced cells plotted against
+estimated forward scatter (FS Lin) and granularity, estimated side
-  mVenus, R10 comprised transduced cells, by detecting mVenus expression. </span><span
+scatter (SS Lin) B: Non transduced cells plotted against mVenus
-  lang=EN-US>E</span><span lang=EN-US style='font-weight:normal'>: Overlay of
+(Analytical gate was set such that 1% or fewer of negative control
-  non-transduced (red) and transduced (green). </span><span lang=EN-US>F</span><span
+cells fell within the positive region (R5).C: Gating transduced cells
-  lang=EN-US style='font-weight:normal'>: Gating non transduced cells
+(R2 </span><span
-  (control). </span><span lang=EN-US>G</span><span lang=EN-US style='font-weight:
+style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
-  normal'>: Non-transduced cells plotted against mVenus (R5). </span><span
+lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R14)
-  lang=EN-US>H</span><span lang=EN-US style='font-weight:normal'>: Gating
+(used plasmids for transfection: pGOI: </span><span lang="EN-US">pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-  transduced cells (R14 </span><span lang=EN-US style='font-family:"Cambria Math","serif";
+(pSB1C3_mVenus: </span><span lang="EN-US">BBa_K404119</span><span
-  font-weight:normal'>&#8793;</span><span lang=EN-US style='font-weight:normal'>R2)
+style="color: rgb(0, 176, 80); font-weight: normal;" lang="EN-US">)</span><span
-  (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span
+style="font-weight: normal;" lang="EN-US">, pHelper, pRC. </span><span
-  lang=EN-US>I</span><span lang=EN-US style='font-weight:normal'>: Transduced
+lang="EN-US">D</span><span style="font-weight: normal;" lang="EN-US">:
-  cells plotted against mVenus, R10 comprised transduced cells, by detecting
+Transduced cells plotted against mVenus, R10 comprised transduced
-  mVenus expression.</span><span lang=EN-US> J</span><span lang=EN-US
+cells, by detecting mVenus expression. </span><span lang="EN-US">E</span><span
-  style='font-weight:normal'>: Overlay of non-transduced (red) and transduced
+style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
-  (green) cells plotted against mVenus expression. </span></p>
+(red) and transduced (green). </span><span lang="EN-US">F</span><span
-  </td>
+style="font-weight: normal;" lang="EN-US">: Gating non transduced
- </tr>
+cells (control). </span><span lang="EN-US">G</span><span
+style="font-weight: normal;" lang="EN-US">: Non-transduced cells
+plotted against mVenus (R5). </span><span lang="EN-US">H</span><span
+style="font-weight: normal;" lang="EN-US">: Gating transduced cells
+(R14 </span><span
+style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;"
+lang="EN-US">≙</span><span style="font-weight: normal;" lang="EN-US">R2)
+(used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span
+lang="EN-US">I</span><span style="font-weight: normal;" lang="EN-US">:
+Transduced cells plotted against mVenus, R10 comprised transduced
+cells, by detecting mVenus expression.</span><span lang="EN-US"> J</span><span
+style="font-weight: normal;" lang="EN-US">: Overlay of non-transduced
+(red) and transduced (green) cells plotted against mVenus expression. </span></p>
+</td>
+</tr>
+</tbody>
 </table>
+<p class="MsoNormal" style="text-indent: 0cm;"><span lang="EN-US">&nbsp;</span></p>
-<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
+<p class="MsoNormal"><span lang="EN-US">&nbsp;</span></p>
+<table class="MsoTableGrid"
-<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
+style="border: medium none ; border-collapse: collapse;" border="1"
+cellpadding="0" cellspacing="0">
-<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
+<tbody>
- style='border-collapse:collapse;border:none'>
+<tr>
- <tr>
+<td
-  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
+style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
-  padding:0cm 5.4pt 0cm 5.4pt'>
+valign="top" width="641">
-  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
+<p class="MsoNormal"
-cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
+style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><span
-  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
+lang="EN-US">&nbsp;</span></p>
-  text-align:center;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
+<p class="MsoNormal"
-  width=541 height=396 id="Diagramm 90"
+style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
-  src="Freiburg10_Modularization_GOI-Dateien/image018.gif"></p>
+align="center"><img id="Diagramm 90"
-  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784852"><span
+src="Freiburg10_Modularization_GOI-Dateien/image018.gif" height="396"
-  lang=EN-US>Figure </span></a><span lang=EN-US>13</span><span lang=EN-US>:
+width="541"></p>
-  Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared
+<p class="MsoCaption" style="text-indent: 0cm;"><a
-  to standard plasmid provided by Stratagene. </span><span lang=EN-US
+name="_Ref275784852"><span lang="EN-US">Figure </span></a><span
-  style='font-weight:normal'>Fluorescence of the standard plasmid pAAV_mVenus (Stratagene)
+lang="EN-US">13</span><span lang="EN-US">: Flow cytometry analysis of
-  and the recombinant pSB1C3_mVenus (BBa_K404119) construct was measured. As
+reassembled vectorplasmid (BBa_K404119) compared to standard plasmid
-  demonstrated mVenus expression is enhanced in the assembled plasmid
+provided by Stratagene. </span><span style="font-weight: normal;"
-  (pSB1C3_mVenus) compared to the standard pAAV_mVenus construct.</span></p>
+lang="EN-US">Fluorescence of the standard plasmid pAAV_mVenus
-  </td>
+(Stratagene) and the recombinant pSB1C3_mVenus (BBa_K404119) construct
- </tr>
+was measured. As demonstrated mVenus expression is enhanced in the
+assembled plasmid (pSB1C3_mVenus) compared to the standard pAAV_mVenus
+construct.</span></p>
+</td>
+</tr>
+</tbody>
 </table>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800689"><span
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800689"><span
+lang="EN-US"><span
-lang=EN-US><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
-</span></span><span lang=EN-US>Conclusion</span></a></h3>
+lang="EN-US">Conclusion</span></a></h3>
+<p class="MsoNormal"><span lang="EN-US">Idea of the modular ‘Virus
-<p class=MsoNormal><span lang=EN-US>Idea of the modular ‘Virus Construction
+Construction
-Kit’ is to provide all required elements for producing recombinant, functional
+Kit’ is to provide all required elements for producing recombinant,
-virus particles delivering encapsidated genes of interest to specific cells.
+functional
-First step was  to modify and modularize the vectorplasmid comprising basically
+virus particles delivering encapsidated genes of interest to specific
-the cis-elements for replication (ITRs), a strong eukaryotic or tissue specific
+cells.
-promoter (pCMV or phTERT), the gene of interest (fluorescent proteins or
+First step was to modify and modularize the vectorplasmid comprising
-suicide genes) and the hGH termination signal. Each element was successfully
+basically
-cloned and reassembled resulting in functional vectorplasmids determined by flow
+the cis-elements for replication (ITRs), a strong eukaryotic or tissue
-cytometry and fluorescence microscopy analyses. Experiments have been performed
+specific
-with mVenus since measurement of fluorescent proteins can be easily performed
+promoter (pCMV or phTERT), the gene of interest (fluorescent proteins
-and visualized. Considering the results, the iGEM team Freiburg_Bioware 2010
+or
-then tested the construct containing the suicide genes thymidine kinase and
+suicide genes) and the hGH termination signal. Each element was
-cytosine deaminase. Further details demonstrating efficient tumor killing,
+successfully
-using prodrug-activating systems, see results page ‘Arming – Killing the
+cloned and reassembled resulting in functional vectorplasmids
+determined by flow
+cytometry and fluorescence microscopy analyses. Experiments have been
+performed
+with mVenus since measurement of fluorescent proteins can be easily
+performed
+and visualized. Considering the results, the iGEM team Freiburg_Bioware
+then tested the construct containing the suicide genes thymidine kinase
+and
+cytosine deaminase. Further details demonstrating efficient tumor
+killing,
+using prodrug-activating systems, see results page ‘Arming – Killing
+the
 tumor’. </span></p>
+<h3 style="margin-left: 0cm; text-indent: 0cm;"><a name="_Toc275800690"><span
-<h3 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275800690"><span
+lang="EN-US"><span
-lang=EN-US><span style='font:7.0pt "Times New Roman"'>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+style="font-family: &quot;Times New Roman&quot;; font-style: normal; font-variant: normal; font-weight: normal; font-size: 7pt; line-height: normal; font-size-adjust: none; font-stretch: normal;"></span></span><span
-</span></span><span lang=EN-US>References</span></a></h3>
+lang="EN-US">References</span></a></h3>
+<p style="text-indent: 36pt;"><span
-<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-font-family:"Calibri","sans-serif"'>Danckwardt, S., Hentze, M.W. &amp; Kulozik,
+lang="EN-US">Danckwardt, S., Hentze, M.W. &amp; Kulozik,
-A.E., 2008. 3' end mRNA processing: molecular mechanisms and implications for
+A.E., 2008. 3' end mRNA processing: molecular mechanisms and
-health and disease. <i>The EMBO journal</i>, 27(3), 482-98. Available at:
+implications for
+health and disease. <i>The EMBO journal</i>, 27(3), 482-98. Available
+at:
 http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
+<p style="text-indent: 36pt;"><span
-<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-font-family:"Calibri","sans-serif"'>Dong, J.Y., Fan, P.D. &amp; Frizzell, R.a.,
+lang="EN-US">Dong, J.Y., Fan, P.D. &amp; Frizzell, R.a.,
 . Quantitative analysis of the packaging capacity of recombinant
-adeno-associated virus. <i>Human gene therapy</i>, 7(17), 2101-12. Available
+adeno-associated virus. <i>Human gene therapy</i>, 7(17), 2101-12.
+Available
 at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
+<p style="text-indent: 36pt;"><span
-<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-font-family:"Calibri","sans-serif"'>Millevoi, S. et al., 2006. An interaction
+lang="EN-US">Millevoi, S. et al., 2006. An interaction
 between U2AF 65 and CF I(m) links the splicing and 3' end processing
 machineries. <i>The EMBO journal</i>, 25(20), 4854-64. Available at:
 http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p>
+<p style="text-indent: 36pt;"><span
-<p style='text-indent:36.0pt'><span lang=EN-US style='font-size:10.0pt;
+style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
-font-family:"Calibri","sans-serif"'>Nott, A., Meislin, S.H. &amp; Moore, M.J.,
+lang="EN-US">Nott, A., Meislin, S.H. &amp; Moore, M.J.,
-. A quantitative analysis of intron effects on mammalian gene expression. <i>RNA
+. A quantitative analysis of intron effects on mammalian gene
+expression. <i>RNA
 (New York, N.Y.)</i>, 9(5), 607-17. Available at:
 http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
+<p style="text-indent: 36pt;"><span lang="EN-US">&nbsp;</span></p>
-<p style='text-indent:36.0pt'><span lang=EN-US>&nbsp;</span></p>
 </html>