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

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Contents

Modularization GOI/Vector Plasmid. 1

Introduction to Modularization. 1

Recombinant and Modular Vector Plasmid 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. 13

Modularization GOI/Vector Plasmid

Introduction to Modularization

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 Vector Plasmid 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).


Left ITR (BBa_K404100)	pCMV (BBa_K404102)	Beta-globin intron (BBa_K404107)	mVenus (BBa_I757008)	hGH terminator (BBa_K404108)	Right ITR (BBa_K404101)
	phTERT (BBa_K404106)		mCherry (BBa_J06504)
			Cytosine deaminase (BBa_K404112)
			mGMK_SR39 (BBa_K404315)
			mGMK_TK30 (BBa_K404113)

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 ).

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 2: 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 3: 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 4: 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 5: 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 6: 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 7: 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 8: 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 9: 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 10: 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 11: 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 12: 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.

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), pp.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), pp.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), pp.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), pp.607-17. Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.

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- href="#_Toc275794994">Recombinant
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+<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275796902"><span
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-</span><span
+style='color:windowtext;display:none;text-decoration:none'> </span><span
- style="color: windowtext; display: none; text-decoration: none;">1</span></a></span></p>
+style='color:windowtext;display:none;text-decoration:none'>2</span></a></span></p>
-<p class="MsoToc3"><span class="MsoHyperlink"><a
- href="#_Toc275794995">Cloning and
+<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275796903"><span
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- style="color: windowtext; display: none; text-decoration: none;">.
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
-</span><span
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- style="color: windowtext; display: none; text-decoration: none;">2</span></a></span></p>
-<p class="MsoToc3"><span class="MsoHyperlink"><a
+<p class=MsoToc3><span class=MsoHyperlink><a href="#_Toc275796904"><span
- href="#_Toc275794996">Testing
+lang=EN-US>Testing functionality of Assembled Vectorplasmid</span><span
-functionality of Assembled Vectorplasmid<span
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
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-</span><span
- style="color: windowtext; display: none; text-decoration: none;">3</span></a></span></p>
+<p class=MsoToc4><span class=MsoHyperlink><a href="#_Toc275796905"><span
-<p class="MsoNormal">&nbsp;</p>
+lang=EN-US>Fluorescence Microscopy of Target Cells Demonstrates GOI Expression</span><span
-<p class="MsoNormal">&nbsp;</p>
+style='color:windowtext;display:none;text-decoration:none'>. </span><span
-<p class="MsoNormal">&nbsp;</p>
+style='color:windowtext;display:none;text-decoration:none'>7</span></a></span></p>
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-GOI/Vector Plasmid</a></h2>
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+lang=EN-US>Analysis of Target Cells by Flow Cytometry demonstrates GOI
-Modularization</a></h3>
+Expression</span><span style='color:windowtext;display:none;text-decoration:
-<p class="MsoNormal">Producing recombinant virus particles
+none'>. </span><span
-for therapeutical
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-quantification assays
+<p class=MsoToc5><span class=MsoHyperlink><a href="#_Toc275796907">Influence of
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-cell for
+</div>
-regulation of transgene expression. In addition to that, the plasmid
-coding for
+</div>
-the Rep and Cap proteins (pRC) can be provided <i>in trans</i>
-leading to a layer
+<h3><a name="_Toc275796901"><span lang=EN-US>Introduction to Modularization</span></a></h3>
-of specificity due to the fact that the two genes are not packaged into
-the
+<p class=MsoNormal><span lang=EN-US>Producing recombinant virus particles for
-capsid since lacking of the ITRs impairs encapsidation. Another
+therapeutical means is, besides specifically target cells, purification and
-advantage of
+quantification assays of AAV-2, one intention of the Virus Construction Kit
-the Helper Free System can be attributed to cotransfection of another
+provided by the iGEM team Freiburg_Bioware 2010. For obtaining a modular
-helper
+toolkit, the complex components of AAV-2 were extracted and redesigned to match
-plasmid (pHelper), which provides the necessary proteins normally
+the iGEM standard. Functional activity was tested in cell culture.</span></p>
-obtained by
-superinfection with helper viruses such as adenovirus or herpes simplex
+<p class=MsoNormal><span lang=EN-US>Differing from the wildtype AAV-2 genome,
-virus.
+the Helper Free System provided by Stratagene comprises three plasmids and a
-These helper genes are required for full viral assembly by regulating
+specialized production cell line. AAV-293 cells derived from the HEK cell line
-gene
+express the stably integrated E1A and E1B helper proteins for efficient virus
-expression of Rep and Cap proteins.</p>
+production. The plasmid containing the inverted terminal repeats (ITRs) is
-<h3><a name="_Toc275794994">Recombinant and Modular
+encapsidated into the preformed capsids after production of single-stranded DNA
-Vector Plasmid Carrying GOI</a></h3>
+therefore also known as vectorplasmid (pGOI). Promoter, <i>beta-globin</i>
-<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
+intron and the hGH terminator signal are flanked by the ITRs and serve in the
-provides a modular Virus
+host cell for regulation of transgene expression. In addition to that, the
-Construction Kit for therapeutical applications, quantification assays
+plasmid coding for the Rep and Cap proteins (pRC) can be provided <i>in trans</i>
-and
+leading to a layer of specificity due to the fact that the two genes are not
-purification approaches depending on capsid modifications and the gene
+packaged into the capsid since lacking of the ITRs impairs encapsidation. Another
-of interest
+advantage of the Helper Free System can be attributed to cotransfection of
-flanked by the inverted terminal repeats (ITRs. In order to produce
+another helper plasmid (pHelper), which provides the necessary proteins
-BioBrick-compatible standardized biological parts, we reengineered the
+normally obtained by superinfection with helper viruses such as adenovirus or
-plasmids
+herpes simplex virus. These helper genes are required for full viral assembly
-and added new components for gene therapy approaches and analysis of
+by regulating gene expression of Rep and Cap proteins.</span></p>
-biological
-activity of assembled BioBrick parts. Each element required for intact
+<h3><a name="_Toc275796902"><span lang=EN-US>Recombinant and Modular Vector Plasmid
-and
+Carrying GOI</span></a></h3>
-functional plasmids comprising the ITRs, a promoter, a putative
-enhancer
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware 2010 provides
-element and the hGH terminator was PCR amplified and fused together <i>de
+a modular Virus Construction Kit for therapeutical applications, quantification
-novo</i>.
+assays and purification approaches depending on capsid modifications and the
-As shown in Figure 1, the vectorplasmid was assembled with the produced
+gene of interest flanked by the inverted terminal repeats (ITRs. In order to
-BioBricks consisting of the left and right ITR (BBa_K404100 and
+produce BioBrick-compatible standardized biological parts, we reengineered the
-BBa_K404101), a
+plasmids and added new components for gene therapy approaches and analysis of
-promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the beta-globin
+biological activity of assembled BioBrick parts. Each element required for
-intron
+intact and functional plasmids comprising the ITRs, a promoter, a putative
-(BBa_K404107), the gene of interests (fluorescent proteins mVenus:
+enhancer element and the hGH terminator was PCR amplified and fused together <i>de
-BBa_I757008 and
+novo</i>.</span><span lang=EN-US> As shown in </span><span
-mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112, mGMK_SR39:
+lang=EN-US>Figure 1</span><span lang=EN-US>, the vectorplasmid was assembled
-BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).</p>
+with the produced BioBricks consisting of the left and right ITR (BBa_K404100
-<table class="MsoNormalTable"
+and BBa_K404101), a promoter (pCMV :BBa_K404102 or phTERT: BBa_K404106)) , the
-  style="border-collapse: collapse;" border="0"
+beta-globin intron (BBa_K404107), the gene of interests (fluorescent proteins
-  cellpadding="0" cellspacing="0">
+mVenus: BBa_I757008 and mCherry: BBa_J06504, suicide genes mGMK_TK30: BBa_K404112,
-  <tbody>
+mGMK_SR39: BBa_K404315 and CD: BBa_K404112) and the hGH terminator (BBa_K404108).</span></p>
-    <tr>
-      <td
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 81.95pt;"
+  style='border-collapse:collapse;border:none'>
- width="109">
+  <tr>
-      <p class="MsoNormal"
+  <td width=109 style='width:81.95pt;border:solid windowtext 1.0pt;padding:
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+cm 5.4pt 0cm 5.4pt'>
- align="center"><img
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- src="Freiburg10_Modularization_GOI_02_files/image001.gif"
+  text-align:center;text-indent:0cm;line-height:normal'><img width=102
- alt="Description: Description: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png"
+  height=83 id="Grafik 1"
- height="83" width="102"></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image001.gif"
-      </td>
+  alt="Beschreibung: http://partsregistry.org/wiki/images/b/ba/Freiburg10_VectorplasmidBrick_1.png"></p>
-      <td
+  </td>
- 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: 76.9pt;"
+  <td width=103 style='width:76.9pt;border:solid windowtext 1.0pt;border-left:
- width="103">
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"><img
+.0pt'><img width=86 height=69 id="Grafik 51"
- src="Freiburg10_Modularization_GOI_02_files/image002.gif"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image002.gif"></span></p>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image002.gif"
+  </td>
- height="69" width="86"></span></p>
+  <td width=122 style='width:91.7pt;border:solid windowtext 1.0pt;border-left:
-      </td>
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
-      <td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- 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: 91.7pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><img width=116
- width="122">
+  height=78 id="Grafik 6"
-      <p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image003.gif"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  alt="Beschreibung: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png"></p>
- align="center"><img
+  </td>
- src="Freiburg10_Modularization_GOI_02_files/image003.gif"
+  <td width=98 style='width:73.75pt;border:solid windowtext 1.0pt;border-left:
- alt="Description: Description: http://partsregistry.org/wiki/images/1/1e/Freiburg10_VectorplasmidBricks_4.png"
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
- height="78" width="116"></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=90
-      <td
+  height=66 id="Grafik 26"
- 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: 73.75pt;"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image004.gif"></p>
- width="98">
+  </td>
-      <p class="MsoNormal"
+  <td width=104 style='width:77.75pt;border:solid windowtext 1.0pt;border-left:
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
- align="center"><img
+  <p class=MsoNormal align=left style='margin-bottom:0cm;margin-bottom:.0001pt;
- src="Freiburg10_Modularization_GOI_02_files/image004.gif"
+  text-align:left;text-indent:0cm;line-height:normal'><img width=96 height=80
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image004.gif"
+  id="Grafik 8" src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image005.gif"
- height="66" width="90"></p>
+  alt="Beschreibung: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png"></p>
-      </td>
+  </td>
-      <td
+  <td width=105 style='width:79.05pt;border:solid windowtext 1.0pt;border-left:
- 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: 77.75pt;"
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
- width="104">
+  <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=98
- style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+  height=79 id="Grafik 4"
- align="left"><img
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image006.gif"
- src="Freiburg10_Modularization_GOI_02_files/image005.gif"
+  alt="Beschreibung: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png"></p>
- alt="Description: Description: http://partsregistry.org/wiki/images/0/06/Freiburg10_VectorplasmidBricks_5.png"
+  </td>
- height="80" width="96"></p>
+ </tr>
-      </td>
+ <tr>
-      <td
+  <td width=109 style='width:81.95pt;border:solid windowtext 1.0pt;border-top:
- 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: 79.05pt;"
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
- width="105">
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <p class="MsoNormal"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+.0pt'><img width=79 height=80 id="Picture 43"
- align="center"><img
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image007.jpg"></span></p>
- src="Freiburg10_Modularization_GOI_02_files/image006.gif"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- alt="Description: Description: http://partsregistry.org/wiki/images/1/18/Freiburg10_VectorplasmidBricks_2.png"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- height="79" width="98"></p>
+.0pt'>Left ITR </span></p>
-      </td>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-    </tr>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-    <tr>
+.0pt'>(</span><span style='font-size:9.0pt;color:black'>BBa_K404100</span><span
-      <td
+  style='font-size:9.0pt'>)</span></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: 81.95pt;"
+  </td>
- width="109">
+  <td width=103 style='width:76.9pt;border-top:none;border-left:none;
-      <p class="MsoNormal"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- align="center"><span style="font-size: 9pt;"><img
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- src="Freiburg10_Modularization_GOI_02_files/image007.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image007.jpg"
+.0pt'><img width=81 height=80 id="Picture 70"
- height="80" width="79"></span></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image008.jpg"></span></p>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'>pCMV</span></p>
- lang="DE">Left ITR </span></p>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <p class="MsoNormal"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+.0pt'>(BBa_K404102)</span></p>
- align="center"><span style="font-size: 9pt;"
+  </td>
- lang="DE">(<span style="color: black;">BBa_K404100</span>)</span></p>
+  <td width=122 style='width:91.7pt;border-top:none;border-left:none;
-      </td>
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-      <td
+  padding:0cm 5.4pt 0cm 5.4pt'>
- 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: 76.9pt;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- width="103">
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <p class="MsoNormal"
+.0pt'><img width=78 height=79 id="Picture 61"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image009.jpg"></span></p>
- align="center"><span style="font-size: 9pt;"><img
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- src="Freiburg10_Modularization_GOI_02_files/image008.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><i><span
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image008.jpg"
+  style='font-size:9.0pt'>Beta-globin</span></i><span style='font-size:9.0pt'>
- height="80" width="80"></span></p>
+  intron </span></p>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'>(</span><span style='font-size:9.0pt;color:black'>BBa_K404107</span><span
- lang="DE">pCMV</span></p>
+  style='font-size:9.0pt'>)</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=98 style='width:73.75pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- lang="DE">(BBa_K404102)</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      </td>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <td
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- 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: 91.7pt;"
+.0pt'><img width=79 height=79 id="Picture 58"
- width="122">
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image010.jpg"></span></p>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"><img
+.0pt'>mVenus (BBa_I757008)</span></p>
- src="Freiburg10_Modularization_GOI_02_files/image009.jpg"
+  </td>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image009.jpg"
+  <td width=104 style='width:77.75pt;border-top:none;border-left:none;
- height="79" width="78"></span></p>
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-      <p class="MsoNormal"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- 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"><i><span style="font-size: 9pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- lang="DE">Beta-globin</span></i><span
+.0pt'><img width=79 height=80 id="Picture 64"
- style="font-size: 9pt;" lang="DE"> intron </span></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image011.jpg"></span></p>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'>hGH terminator</span></p>
- lang="DE">(<span style="color: black;">BBa_K404107</span>)</span></p>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      </td>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <td
+.0pt'>(BBa_K404108)</span></p>
- 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: 73.75pt;"
+  </td>
- width="98">
+  <td width=105 style='width:79.05pt;border-top:none;border-left:none;
-      <p class="MsoNormal"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- align="center"><span style="font-size: 9pt;"><img
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- src="Freiburg10_Modularization_GOI_02_files/image010.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image010.jpg"
+.0pt'><img width=78 height=80 id="Picture 46"
- height="79" width="79"></span></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image012.jpg"></span></p>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'>Right ITR</span></p>
- lang="DE">mVenus (BBa_I757008)</span></p>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      </td>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <td
+.0pt'>(BBa_K404101)</span></p>
- 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: 77.75pt;"
+  </td>
- width="104">
+ </tr>
-      <p class="MsoNormal"
+ <tr>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=109 style='width:81.95pt;border:solid windowtext 1.0pt;border-top:
- align="center"><span style="font-size: 9pt;"><img
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
- src="Freiburg10_Modularization_GOI_02_files/image011.jpg"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image011.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- height="80" width="80"></span></p>
+.0pt'>&nbsp;</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=103 style='width:76.9pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- lang="DE">hGH terminator</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'><img width=79 height=80 id="Picture 67"
- lang="DE">(BBa_K404108)</span></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image013.jpg"></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'><span style='font-size:
- 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: 79.05pt;"
+.0pt'>phTERT (BBa_K404106)</span></p>
- width="105">
+  </td>
-      <p class="MsoNormal"
+  <td width=122 style='width:91.7pt;border-top:none;border-left:none;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- align="center"><span style="font-size: 9pt;"><img
+  padding:0cm 5.4pt 0cm 5.4pt'>
- src="Freiburg10_Modularization_GOI_02_files/image012.jpg"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image012.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- height="79" width="79"></span></p>
+.0pt'>&nbsp;</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=98 style='width:73.75pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- lang="DE">Right ITR</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'><img width=79 height=79 id="Picture 109"
- lang="DE">(BBa_K404101)</span></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image014.jpg"></span></p>
-      </td>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-    </tr>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-    <tr>
+.0pt'>mCherry</span></p>
-      <td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- width="109">
+.0pt'>(BBa_J06504)</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=104 style='width:77.75pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- lang="DE">&nbsp;</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      </td>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <td
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- 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: 76.9pt;"
+.0pt'>&nbsp;</span></p>
- width="103">
+  </td>
-      <p class="MsoNormal"
+  <td width=105 style='width:79.05pt;border-top:none;border-left:none;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- align="center"><span style="font-size: 9pt;"><img
+  padding:0cm 5.4pt 0cm 5.4pt'>
- src="Freiburg10_Modularization_GOI_02_files/image013.jpg"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image013.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- height="80" width="79"></span></p>
+.0pt'>&nbsp;</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+ </tr>
- align="center"><span style="font-size: 9pt;"
+ <tr>
- lang="DE">phTERT (BBa_K404106)</span></p>
+  <td width=109 style='width:81.95pt;border:solid windowtext 1.0pt;border-top:
-      </td>
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
-      <td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- 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: 91.7pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- width="122">
+.0pt'>&nbsp;</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=103 style='width:76.9pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- lang="DE">&nbsp;</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      </td>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <td
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- 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: 73.75pt;"
+.0pt'>&nbsp;</span></p>
- width="98">
+  </td>
-      <p class="MsoNormal"
+  <td width=122 style='width:91.7pt;border-top:none;border-left:none;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- align="center"><span style="font-size: 9pt;"><img
+  padding:0cm 5.4pt 0cm 5.4pt'>
- src="Freiburg10_Modularization_GOI_02_files/image014.jpg"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image014.jpg"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- height="79" width="79"></span></p>
+.0pt'>&nbsp;</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=98 style='width:73.75pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- lang="DE">mCherry</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'><img width=79 height=81 id="Picture 10"
- lang="DE">(BBa_J06504)</span></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image015.jpg"></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'><span style='font-size:
- 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: 77.75pt;"
+.0pt'>Cytosine deaminase (BBa_K404112)</span></p>
- width="104">
+  </td>
-      <p class="MsoNormal"
+  <td width=104 style='width:77.75pt;border-top:none;border-left:none;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- align="center"><span style="font-size: 9pt;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- lang="DE">&nbsp;</span></p>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      </td>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <td
+.0pt'>&nbsp;</span></p>
- 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: 79.05pt;"
+  </td>
- width="105">
+  <td width=105 style='width:79.05pt;border-top:none;border-left:none;
-      <p class="MsoNormal"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- align="center"><span style="font-size: 9pt;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- lang="DE">&nbsp;</span></p>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      </td>
+.0pt'>&nbsp;</span></p>
-    </tr>
+  </td>
-    <tr>
+ </tr>
-      <td
+ <tr>
- style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+  <td width=109 style='width:81.95pt;border:solid windowtext 1.0pt;border-top:
- width="109">
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'>&nbsp;</span></p>
- lang="DE">&nbsp;</span></p>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      </td>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <td
+.0pt'>&nbsp;</span></p>
- 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: 76.9pt;"
+  </td>
- width="103">
+  <td width=103 style='width:76.9pt;border-top:none;border-left:none;
-      <p class="MsoNormal"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- align="center"><span style="font-size: 9pt;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- lang="DE">&nbsp;</span></p>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      </td>
+.0pt'>&nbsp;</span></p>
-      <td
+  </td>
- 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: 91.7pt;"
+  <td width=122 style='width:91.7pt;border-top:none;border-left:none;
- width="122">
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-      <p class="MsoNormal"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- 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"><span style="font-size: 9pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- lang="DE">&nbsp;</span></p>
+.0pt'>&nbsp;</span></p>
-      </td>
+  </td>
-      <td
+  <td width=98 style='width:73.75pt;border-top:none;border-left:none;
- 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: 73.75pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- width="98">
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"><img
+.0pt'><img width=79 height=80 id="Picture 13"
- src="Freiburg10_Modularization_GOI_02_files/image015.jpg"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image016.jpg"></span></p>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image015.jpg"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- height="81" width="80"></span></p>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <p class="MsoNormal"
+.0pt'>mGMK_SR39</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"><span style="font-size: 9pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- lang="DE">Cytosine deaminase (BBa_K404112)</span></p>
+.0pt'>(BBa_K404315)</span></p>
-      </td>
+  </td>
-      <td
+  <td width=104 style='width:77.75pt;border-top:none;border-left:none;
- 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: 77.75pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- width="104">
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'>&nbsp;</span></p>
- lang="DE">&nbsp;</span></p>
+  </td>
-      </td>
+  <td width=105 style='width:79.05pt;border-top:none;border-left:none;
-      <td
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- 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: 79.05pt;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- width="105">
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <p class="MsoNormal"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+.0pt'>&nbsp;</span></p>
- align="center"><span style="font-size: 9pt;"
+  </td>
- lang="DE">&nbsp;</span></p>
+ </tr>
-      </td>
+ <tr>
-    </tr>
+  <td width=109 style='width:81.95pt;border:solid windowtext 1.0pt;border-top:
-    <tr>
+  none;padding:0cm 5.4pt 0cm 5.4pt'>
-      <td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- width="109">
+.0pt'>&nbsp;</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=103 style='width:76.9pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- lang="DE">&nbsp;</span></p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-      <p class="MsoNormal"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- align="center"><span style="font-size: 9pt;"
+.0pt'>&nbsp;</span></p>
- lang="DE">&nbsp;</span></p>
+  </td>
-      </td>
+  <td width=122 style='width:91.7pt;border-top:none;border-left:none;
-      <td
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- 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: 76.9pt;"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- width="103">
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <p class="MsoNormal"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+.0pt'>&nbsp;</span></p>
- align="center"><span style="font-size: 9pt;"
+  </td>
- lang="DE">&nbsp;</span></p>
+  <td width=98 style='width:73.75pt;border-top:none;border-left:none;
-      </td>
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-      <td
+  padding:0cm 5.4pt 0cm 5.4pt'>
- 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: 91.7pt;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- width="122">
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <p class="MsoNormal"
+.0pt'><img width=81 height=81 id="Picture 7"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image017.jpg"></span></p>
- align="center"><span style="font-size: 9pt;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- lang="DE">&nbsp;</span></p>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      </td>
+.0pt'>mGMK_TK30</span></p>
-      <td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- 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: 73.75pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
- width="98">
+.0pt'>(BBa_K404113)</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=104 style='width:77.75pt;border-top:none;border-left:none;
- align="center"><span style="font-size: 9pt;"><img
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- src="Freiburg10_Modularization_GOI_02_files/image016.jpg"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image016.jpg"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- height="79" width="80"></span></p>
+  text-align:center;text-indent:0cm;line-height:normal'><span style='font-size:
-      <p class="MsoNormal"
+.0pt'>&nbsp;</span></p>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  </td>
- align="center"><span style="font-size: 9pt;"
+  <td width=105 style='width:79.05pt;border-top:none;border-left:none;
- lang="DE">mGMK_SR39</span></p>
+  border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
-      <p class="MsoNormal"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- 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"><span style="font-size: 9pt;"
+  text-align:center;text-indent:0cm;line-height:normal;page-break-after:avoid'><span
- lang="DE">(BBa_K404315)</span></p>
+  style='font-size:9.0pt'>&nbsp;</span></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: 77.75pt;"
+ <tr>
- width="104">
+  <td width=641 colspan=6 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-align: center; text-indent: 0cm; line-height: normal;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- align="center"><span style="font-size: 9pt;"
+  text-align:center;text-indent:0cm;line-height:normal;page-break-after:avoid'><span
- lang="DE">&nbsp;</span></p>
+  style='font-size:9.0pt'><img width=555 height=116 id="Grafik 2057"
-      </td>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image018.gif"></span></p>
-      <td
+  </td>
- 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: 79.05pt;"
+ </tr>
- width="105">
+ <tr>
-      <p class="MsoNormal"
+  <td width=641 colspan=6 style='width:481.1pt;border:solid windowtext 1.0pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
- align="center"><span style="font-size: 9pt;"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275644946"><span
- lang="DE">&nbsp;</span></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>1</span><span lang=EN-US>: </span><span
-      </td>
+  lang=EN-US style='font-weight:normal'>Overview of the theoretical sequence of
-    </tr>
+  each BioBrick provided within the Virus Construction Kit for an intact and
-    <tr>
+  fully functional rAAV genome. The plasmid in the lowest panel was used for
-      <td
+  tumor killing in combination with plasmids coding for modified capsid
- style="border-style: none solid solid; border-color: -moz-use-text-color windowtext windowtext; border-width: medium 1pt 1pt; padding: 0cm 5.4pt; width: 81.95pt;"
+  proteins. More detailed infomartion about these constructs can be found under
- width="109">
+  ‘Arming: Killing the tumor’ and ‘N-terminal fusion for Targeting’.</span></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+ </tr>
- align="center"><span style="font-size: 9pt;"
- lang="DE">&nbsp;</span></p>
-      </td>
-      <td
- 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: 76.9pt;"
- width="103">
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
- align="center"><span style="font-size: 9pt;"
- lang="DE">&nbsp;</span></p>
-      </td>
-      <td
- 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: 91.7pt;"
- width="122">
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
- align="center"><span style="font-size: 9pt;"
- lang="DE">&nbsp;</span></p>
-      </td>
-      <td
- 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: 73.75pt;"
- width="98">
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
- align="center"><span style="font-size: 9pt;"><img
- src="Freiburg10_Modularization_GOI_02_files/image017.jpg"
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image017.jpg"
- height="81" width="81"></span></p>
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
- align="center"><span style="font-size: 9pt;"
- lang="DE">mGMK_TK30</span></p>
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
- align="center"><span style="font-size: 9pt;"
- lang="DE">(BBa_K404113)</span></p>
-      </td>
-      <td
- 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: 77.75pt;"
- width="104">
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
- align="center"><span style="font-size: 9pt;"
- lang="DE">&nbsp;</span></p>
-      </td>
-      <td
- 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: 79.05pt;"
- width="105">
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
- align="center"><span style="font-size: 9pt;"
- lang="DE">&nbsp;</span></p>
-      </td>
-    </tr>
-    <tr>
-      <td colspan="6"
- 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;"
- width="641">
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
- align="center"><span style="font-size: 9pt;"><img
- src="Freiburg10_Modularization_GOI_02_files/image018.gif"
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image018.gif"
- height="116" width="555"></span></p>
-      </td>
-    </tr>
-    <tr>
-      <td colspan="6"
- 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;"
- width="641">
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
- name="_Ref275644946">Figure </a>1: <span
- style="font-weight: normal;">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>
-<h3><a name="_Toc275794995">Cloning and Combination
-Strategies for the
+<h3><a name="_Toc275796903"><span lang=EN-US>Cloning and Combination Strategies
-Vectorplasmid</a> </h3>
+for the Vectorplasmid</span></a><span lang=EN-US> </span></h3>
-<p class="MsoNormal">Organization of the recombinant viral
-DNA was modified ensuring
+<p class=MsoNormal><span lang=EN-US>Organization of the recombinant viral DNA was
-several layers of specificity to our systems including a tumor-specific
+modified ensuring several layers of specificity to our systems including a
-promoter and suicide genes encoding prodrug convertases. In order to
+tumor-specific promoter and suicide genes encoding prodrug convertases. In
-modularize
+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
-the rAAV sequence, each plasmid element (Figure 1) was PCR-amplified
+the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team Freiburg_Bioware
-and cloned
+performed three site-directed mutagenesis in the gene of interest TK30 (BBa_K404109)
-into the iGEM standard plasmid pSB1C3. Furthermore, the iGEM team
+and cytosine deaminase (</span><span lang=EN-US style='font-size:9.0pt;
-Freiburg_Bioware 2010 performed three site-directed mutagenesis in the
+line-height:200%'>BBa_K404112</span><span lang=EN-US>) for deletion of PstI and
-gene of
+NgoMIV iGEM site (for further information see the results page of ‘Arming –
-interest TK30 (BBa_K404109) and cytosine deaminase (<span
+Killing the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich
- style="font-size: 9pt; line-height: 200%;">BBa_K404112</span>)
+regions forming T-shaped hairpins during replication, PCR amplification was not
-for deletion of PstI and NgoMIV
+possible. Hence a cloning strategy was developed by the iGEM team Freiburg in
-iGEM site (for further information see the results page of ‘Arming –
+order to provide BioBrick-compatible ITRs (see ).</span></p>
-Killing
-the tumor’). Since the inverted terminal repeats (ITRs) are GC-rich
+<p class=MsoNormal><span lang=EN-US>In </span><span
-regions
+lang=EN-US>Figure 2</span><span lang=EN-US> the schematic overview of the
-forming T-shaped hairpins during replication, PCR amplification was not
+modularization process can be seen which has been followed to conduct the
-possible. Hence a cloning strategy was developed by the iGEM team
+assembly steps required for functional vectorplasmids.</span></p>
-Freiburg in
-order to provide BioBrick-compatible ITRs (see ).</p>
+<div align=center>
-<p class="MsoNormal">In Figure 2 the schematic overview of
-the modularization
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-process can be seen which has been followed to conduct the assembly
+  style='border-collapse:collapse;border:none'>
-steps
+  <tr style='height:26.15pt'>
-required for functional vectorplasmids.</p>
+  <td width=454 valign=top style='width:340.15pt;border:solid windowtext 1.0pt;
-<div align="center">
+  padding:0cm 5.4pt 0cm 5.4pt;height:26.15pt'>
-<table class="MsoNormalTable"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-  style="border-collapse: collapse;" border="0"
+cm;line-height:normal;page-break-after:avoid'><img width=439 height=272
-  cellpadding="0" cellspacing="0">
+  id="Grafik 2" src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image019.gif"
-  <tbody>
+  alt="Beschreibung: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"></p>
-    <tr style="height: 26.15pt;">
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275783119"><span
-      <td
+  lang=EN-US>Figure </span></a><span lang=EN-US>2</span><span lang=EN-US>: </span><span
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 340.15pt; height: 26.15pt;"
+  lang=EN-US style='font-weight:normal'>Assembly procedure for fusion of
- valign="top" width="454">
+  BioBricks and composite parts to a fully assembled and functional plasmid
-      <p class="MsoNormal"
+  coding for your gene of interest. This plasmid can be cotransfected with two
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+  helper plasmids providing protein for assembly and encapsidating of the rAAV
- src="Freiburg10_Modularization_GOI_02_files/image019.gif"
+  genome (your gene of interest) into the capsids.</span></p>
- alt="Description: Description: http://partsregistry.org/wiki/images/1/1c/Freiburg10_Vectorplasmid_cloning.png"
+  </td>
- height="272" width="439"></p>
+ </tr>
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
- name="_Ref275783119">Figure </a>2: <span
- style="font-weight: normal;">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.</span></p>
-      </td>
-    </tr>
-  </tbody>
 </table>
 </div>
-<p class="MsoNormal">&nbsp;</p>
-<p class="MsoNormal">The iGEM team Freiburg_Bioware
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-provides two examples
-demonstrating the assembly procedure for constructing vectorplasmids.
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware provides two
-The first
+examples demonstrating the assembly procedure for constructing vectorplasmids.
-representative example is the fusion of the BioBrick part <i>beta-globin</i>
+The first representative example is the fusion of the BioBrick part <i>beta-globin</i>
-to
+to the composite parts containing the 5´ elements of the plasmids, which are
-the composite parts containing the 5´ elements of the plasmids, which
+left ITR and CMV or phTERT promoter, respectively.</span></p>
-are left
-ITR and CMV or phTERT promoter, respectively.</p>
+<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
-<p class="MsoNormal" style="text-indent: 0cm;">As
+for assembling the BioBricks <i>beta-globin </i>intron and leftITR_CMV together
-shown in Figure 3 the theoretical
+can be observed. </span></p>
-cloning performed for assembling the BioBricks <i>beta-globin </i>intron
-and
+<div align=center>
-leftITR_CMV together can be observed. </p>
-<div align="center">
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-<table class="MsoNormalTable"
+  style='border-collapse:collapse;border:none'>
-  style="border-collapse: collapse;" border="0"
+  <tr style='height:23.95pt'>
-  cellpadding="0" cellspacing="0">
+  <td width=467 valign=top style='width:349.9pt;border:solid windowtext 1.0pt;
-  <tbody>
+  padding:0cm 5.4pt 0cm 5.4pt;height:23.95pt'>
-    <tr style="height: 23.95pt;">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-      <td
+cm;line-height:normal;page-break-after:avoid'><img width=452 height=530
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 349.9pt; height: 23.95pt;"
+  id="Grafik 74"
- valign="top" width="467">
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image020.gif"></p>
-      <p class="MsoNormal"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275783160"><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>3</span><span lang=EN-US>:</span><span
- src="Freiburg10_Modularization_GOI_02_files/image020.gif"
+  lang=EN-US> </span><span lang=EN-US style='font-weight:normal'>Theoretical
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image020.gif"
+  cloning of the composite part leftITR_CMV to the <i>beta-globin</i> intron
- height="530" width="452"></p>
+  BioBrick leading to the plasmid leftITR_CMV_<i>beta-globin</i> intron.</span></p>
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
+  </td>
- name="_Ref275783160">Figure </a>3: <span
+ </tr>
- style="font-weight: normal;">Theoretical cloning of the
-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;">&nbsp;</p>
-<p class="MsoNormal">The plasmids were digested with both
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-XbaI and PstI
-(beta-globin intron: <span
+<p class=MsoNormal><span lang=EN-US>The plasmids were digested with both XbaI
- style="font-size: 9pt; line-height: 200%; color: black;">BBa_K404107</span>)
+and PstI (beta-globin intron: </span><span lang=EN-US style='font-size:9.0pt;
-or SpeI and PstI (leftITR_CMV) and loaded on an agarose gel. As
+line-height:200%;color:black'>BBa_K404107</span><span lang=EN-US>) or SpeI and
-demonstrated in
+PstI (leftITR_CMV) and loaded on an agarose gel. As demonstrated in the
-the preparative gel in Figure 4, the expected bands could be detected
+preparative gel in </span><span lang=EN-US>Figure 4</span><span lang=EN-US>,
-under UV
+the expected bands could be detected under UV light and the extracted DNA could
-light and the extracted DNA could be successfully ligated. Each
+be successfully ligated. Each assembly step for producing BioBrick
-assembly step
+intermediates was conducted following the same strategy.</span></p>
-for producing BioBrick intermediates was conducted following the same
-strategy.</p>
+<div align=center>
-<div align="center">
-<table class="MsoNormalTable"
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-  style="border-collapse: collapse;" border="0"
+  style='border-collapse:collapse;border:none'>
-  cellpadding="0" cellspacing="0">
+  <tr style='height:129.95pt'>
-  <tbody>
+  <td width=417 valign=top style='width:312.75pt;border:solid windowtext 1.0pt;
-    <tr style="height: 129.95pt;">
+  padding:0cm 5.4pt 0cm 5.4pt;height:129.95pt'>
-      <td
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 312.75pt; height: 129.95pt;"
+cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
- valign="top" width="417">
+  <p class=MsoNormal align=right style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <p class="MsoNormal"
+  text-align:right;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p>
+  width=397 height=194 id="Grafik 77"
-      <p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image021.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
+  <p class=MsoCaption align=left style='text-align:left;text-indent:0cm'><span
- src="Freiburg10_Modularization_GOI_02_files/image021.gif"
+  lang=EN-US>Figure </span><span lang=EN-US>4</span><span lang=EN-US>: Assembly
- alt="Description: Description: \\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"
+  intermediate in fusion of the vectorplasmids containing different promoters. </span><span
- height="194" width="397"></p>
+  lang=EN-US style='font-weight:normal'>Fusion of the BioBrick part <i>beta-globin</i>
-      <p class="MsoCaption"
+  (</span><span lang=EN-US style='color:black'>BBa_K404107</span><span
- style="text-align: left; text-indent: 0cm;" align="left">Figure
+  lang=EN-US style='font-weight:normal'>) intron to the composite parts
-: Assembly intermediate in fusion of the vectorplasmids containing
+  leftITR_pCMV and leftITR_phTERT, respectively, was performed following the
-different promoters. <span style="font-weight: normal;">Fusion
+  BioBrick assembly strategy by digesting the insert with PstI and XbaI and the
-of the BioBrick part <i>beta-globin</i> (</span><span
+  vectors with SpeI and PstI. The left lane shows the expected fragment at
- style="color: black;">BBa_K404107</span><span
+  around 560 bp which corresponds to the <i>beta-globin</i> intron fragment, in
- style="font-weight: normal;">) intron to the composite parts
+  contrast to the two lanes in the center and on the right which correspond to
-leftITR_pCMV and leftITR_phTERT, respectively, was performed following
+  linearized plasmids after digesting with above mentioned iGEM restriction
-the BioBrick assembly strategy by digesting the insert with PstI and
+  sites. M, GeneRuler DNA ladder mix; Insert, pSB1C3_<i>beta-globin</i> intron;
-XbaI and the vectors with SpeI and PstI. The left lane shows the
+  Vector pCMV, pSB1C3_leftITR_pCMV; Vector phTERT, pSB1C3_leftITR_phTERT.</span></p>
-expected fragment at around 560 bp which corresponds to the <i>beta-globin</i>
+  </td>
-intron fragment, in contrast to the two lanes in the center and on the
+ </tr>
-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">&nbsp;</p>
-<p class="MsoNormal">Separated fragments were extracted
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-using the Gel Extraction
-Kit provided by Qiagen (Hilden, Germany) and ligated with T4-ligase.
+<p class=MsoNormal><span lang=EN-US>Separated fragments were extracted using
-After
+the Gel Extraction Kit provided by Qiagen (Hilden, Germany) and ligated with
-ligation has been carried out, <i>E. coli</i> XL-1B cells
+T4-ligase. After ligation has been carried out, <i>E. coli</i> XL-1B cells were
-were transformed and incubated
+transformed and incubated over night at 37°C. Picking clones from the
-over night at 37°C. Picking clones from the transformation plate was
+transformation plate was performed the following day and DYT medium was
-performed
+inoculated incubating overnight. Plasmid DNA was isolated and test digestion
-the following day and DYT medium was inoculated incubating overnight.
+revealed that cloning was successful obtaining the composite part leftITR_CMV_<i>beta-globin</i>
-Plasmid
+intron (BBa_K404117).</span></p>
-DNA was isolated and test digestion revealed that cloning was
-successful
+<p class=MsoNormal><span lang=EN-US>Plasmid production incorporating all
-obtaining the composite part leftITR_CMV_<i>beta-globin</i>
+required elements for transgene expression and genome encapsidation into empty
-intron
+viral capsids was performed by fusing the downstream elements consisting of the
-(BBa_K404117).</p>
+hGH terminator and right ITR to the intermediate part providing the gene of
-<p class="MsoNormal">Plasmid production incorporating all
+interest and the promoter fused to the left ITR. </span><span
-required elements for
+lang=EN-US>Figure 5</span><span lang=EN-US> demonstrates the assembly performed
-transgene expression and genome encapsidation into empty viral capsids
+with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus and
-was
+pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after digestion on the
-performed by fusing the downstream elements consisting of the hGH
+left lane fits to the hGH-terminator_rightITR length. The isolated fragments
-terminator
+were ligated and successful assembly was confirmed by test digestion obtaining
-and right ITR to the intermediate part providing the gene of interest
+the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus_hGH_rightITR
-and the
+(</span><span lang=EN-US style='line-height:200%;color:black'>BBa_K404124</span><span
-promoter fused to the left ITR. Figure 5 demonstrates the assembly
+lang=EN-US>). </span></p>
-performed
-with pSB1C3_leftITR_phTERT_<i>beta-globin</i> intron_mVenus
+<div align=center>
-and
-pSB1C3_hGH_rightITR (BBa_K404116). The fragment obtained after
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-digestion on the
+  style='border-collapse:collapse;border:none'>
-left lane fits to the hGH-terminator_rightITR length. The isolated
+ <tr style='height:136.9pt'>
-fragments
+  <td width=411 valign=top style='width:308.4pt;border:solid windowtext 1.0pt;
-were ligated and successful assembly was confirmed by test digestion
+  padding:0cm 5.4pt 0cm 5.4pt;height:136.9pt'>
-obtaining
+  <p class=MsoNormal align=right style='margin-bottom:0cm;margin-bottom:.0001pt;
-the vectorplasmid pSB1C3_leftITR_phTERT_<i>beta-globin</i>
+  text-align:right;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
-intron_mVenus_hGH_rightITR (<span style="color: black;">BBa_K404124</span>).
+  width=397 height=184 id="Grafik 80"
-</p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image022.gif"
-<div align="center">
+  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>
-<table class="MsoNormalTable"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784510"><span
-  style="border-collapse: collapse;" border="0"
+  lang=EN-US>Figure </span></a><span lang=EN-US>5</span><span lang=EN-US
- cellpadding="0" cellspacing="0">
+  style='font-weight:normal'>: </span><span lang=EN-US>Modularization of the
-  <tbody>
+  assembled vectorplasmid containing the phTERT promoter and mVenus as gene of
-    <tr style="height: 136.9pt;">
+  interest.</span><span lang=EN-US style='font-weight:normal'> Fusion of the
-      <td
+  composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus part  to the
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 308.4pt; height: 136.9pt;"
+  composite parts pSB1C3_hGH_rightITR was performed following the BioBrick
- valign="top" width="411">
+  assembly strategy by digesting the insert with XbaI and PstI and the vector
-      <p class="MsoNormal"
+  with SpeI and PstI. The left lane corresponds to linearized plasmid after
- style="margin-bottom: 0.0001pt; text-align: right; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+  digesting with above mentioned iGEM restriction sites whereas the right lane
- align="right"><img
+  reveals an intensive band at around 650 bp confirming the expected size of
- src="Freiburg10_Modularization_GOI_02_files/image022.gif"
+bp of hGH_rITR. M, GeneRuler DNA ladder mix; Vector, pSB1C3_leftITR_phTERT_beta-globin
- alt="Description: Description: \\132.230.232.133\x\users\FreiGem\iGEM2010\Labor\Manual- Virus Construction Kit\Modularization - GOI\18.09_Cloning_Full_phTERT_mVenus.png"
+  intron_mVenus; Insert, pSB1C3_ pSB1C3_hGH_rightITR.</span></p>
- height="184" width="397"></p>
+  </td>
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
+ </tr>
- name="_Ref275784510">Figure </a>5<span
- style="font-weight: normal;">: </span>Modularization
-of the assembled vectorplasmid containing the phTERT promoter and
-mVenus as gene of interest.<span style="font-weight: normal;">
-Fusion of the composite pSB1C3_leftITR_phTERT_beta-globin intron_mVenus
-part&nbsp; 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.</span></p>
-      </td>
-    </tr>
-  </tbody>
 </table>
 </div>
-<p class="MsoNormal" style="text-indent: 0cm;">&nbsp;</p>
-<p class="MsoNormal">Since cloning does not confirm
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-biological activity, we
-analyzed the plasmids and their functional components, hGH terminator
+<p class=MsoNormal><span lang=EN-US>Since cloning does not confirm biological
-and <i>beta-globin</i>
+activity, we analyzed the plasmids and their functional components, hGH
-intron, in cell culture. Assembled plasmids have been cotransfected,
+terminator and <i>beta-globin</i> intron, in cell culture. Assembled plasmids have
-using
+been cotransfected, using AAV-293 cells, which provide the stable integrated
-AAV-293 cells, which provide the stable integrated E1A and E1B genes,
+E1A and E1B genes, with helper plasmids required for capsid assembly  and
-with
+genome encapsidation (pRC and pHelper) in a molar ratio of 1:1:1
-helper plasmids required for capsid assembly&nbsp; and genome
+(pGOI:pRC:pHelper). Virus particles containing the single stranded DNA were
-encapsidation
+harvested 72-hours post transfection and HT1080 cells transduced with constant
-(pRC and pHelper) in a molar ratio of 1:1:1 (pGOI:pRC:pHelper). Virus
+volumes of viral vectors. 48-hours post infection; transduced cells expressing
-particles
+the gene of interest were analyzed by flow cytometry. Facilitating and
-containing the single stranded DNA were harvested 72-hours post
+demonstrating the analysis of functionality of the assembled plasmid, mVenus
-transfection
+was used in first place since fluorescent proteins enable facile visualization
-and HT1080 cells transduced with constant volumes of viral vectors.
+using fluorescent microscopy and flow cytometry analysis.</span></p>
--hours
-post infection; transduced cells expressing the gene of interest were
+<h3><a name="_Toc275796904"><span lang=EN-US>Testing functionality of Assembled
-analyzed
+Vectorplasmid</span></a></h3>
-by flow cytometry. Facilitating and demonstrating the analysis of
-functionality
+<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275796905"><span
-of the assembled plasmid, mVenus was used in first place since
+lang=EN-US>Fluorescence Microscopy of Target Cells Demonstrates GOI Expression</span></a></h4>
-fluorescent
-proteins enable facile visualization using fluorescent microscopy and
+<p class=MsoNormal><span lang=EN-US>Qualitative analysis of mVenus expression
-flow
+by fluorescence microscopy was conducted using Axio Observer Z1 showing that
-cytometry analysis.</p>
+transduced HT1080 cells and non-transduced cells could be easily distinguished.
-<h3><a name="_Toc275794996">Testing functionality of
+In </span><span lang=EN-US>Figure 6</span><span lang=EN-US> cells were excited
-Assembled Vectorplasmid</a></h3>
+with 505nm and fluorescence emission at 536nm was detected. Therefore, successful
-<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
+infection of tumor cells by recombinant viral particles carrying the assembled vectorplasmid
- lang="DE">Fluorescence</span>
+coding for mVenus could be demonstrated. </span></p>
-Microscopy of Target Cells Demonstrates GOI Expression</h4>
-<p class="MsoNormal">Qualitative analysis of mVenus
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-expression by fluorescence
+  style='border-collapse:collapse;border:none'>
-microscopy was conducted using Axio Observer Z1 showing that transduced
+  <tr>
-HT1080
+  <td width=334 valign=top style='width:250.7pt;border:solid windowtext 1.0pt;
-cells and non-transduced cells could be easily distinguished. In Figure
+  padding:0cm 5.4pt 0cm 5.4pt'>
-cells
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-were excited with 505nm and fluorescence emission at 536nm was
+cm;line-height:normal'><span lang=EN-US>A</span></p>
-detected.
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-Therefore, successful infection of tumor cells by recombinant viral
+  text-align:center;text-indent:0cm;line-height:normal'><img width=264
-particles
+  height=218 id="Grafik 18"
-carrying the assembled vectorplasmid coding for mVenus could be
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image023.jpg"
-demonstrated. </p>
+  alt="Beschreibung: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"></p>
-<table class="MsoNormalTable"
+  </td>
-  style="border-collapse: collapse;" border="0"
+  <td width=307 valign=top style='width:230.4pt;border:solid windowtext 1.0pt;
-  cellpadding="0" cellspacing="0">
+  border-left:none;padding:0cm 5.4pt 0cm 5.4pt'>
-  <tbody>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-    <tr>
+cm;line-height:normal'><span lang=EN-US>B</span></p>
-      <td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 250.7pt;"
+  text-align:center;text-indent:0cm;line-height:normal'><img width=242
- valign="top" width="334">
+  height=219 id="Grafik 19"
-      <p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image024.jpg"
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">A</p>
+  alt="Beschreibung: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+ </tr>
- align="center"><img
+ <tr>
- src="Freiburg10_Modularization_GOI_02_files/image023.jpg"
+  <td width=334 valign=top style='width:250.7pt;border:solid windowtext 1.0pt;
- alt="Description: Description: Freiburg10_2Transd30µg_unverd_2_(c1).JPG (1388×1040)"
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
- height="218" width="264"></p>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-      </td>
+cm;line-height:normal'><span lang=EN-US>C</span></p>
-      <td
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- 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;"
+  text-align:center;text-indent:0cm;line-height:normal'><img width=258
- valign="top" width="307">
+  height=195 id="Grafik 16"
-      <p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image025.jpg"
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">B</p>
+  alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  <td width=307 valign=top style='width:230.4pt;border-top:none;border-left:
- align="center"><img
+  none;border-bottom:solid windowtext 1.0pt;border-right:solid windowtext 1.0pt;
- src="Freiburg10_Modularization_GOI_02_files/image024.jpg"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- alt="Description: Description: https://static.igem.org/mediawiki/2010/f/f1/Freiburg10_2Transd30%C2%B5g_unverd_%28c1%29.JPG"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
- height="220" width="242"></p>
+cm;line-height:normal'><span lang=EN-US>D</span></p>
-      </td>
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
-    </tr>
+  text-align:center;text-indent:0cm;line-height:normal'><img width=257
-    <tr>
+  height=194 id="Grafik 17"
-      <td
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image026.jpg"
- 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;"
+  alt="Beschreibung: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"></p>
- valign="top" width="334">
+  </td>
-      <p class="MsoNormal"
+ </tr>
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">C</p>
+ <tr>
-      <p class="MsoNormal"
+  <td width=641 colspan=2 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
- align="center"><img
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784524"><span
- src="Freiburg10_Modularization_GOI_02_files/image025.jpg"
+  lang=EN-US>Figure </span></a><span lang=EN-US>6</span><span lang=EN-US>: </span><span
- alt="Description: Description: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
+  lang=EN-US style='font-weight:normal'>Fluorescence microscopy (Exciatation:
- height="195" width="258"></p>
+nm, Emission: 536nm) was performed for detection of transduced cell
-      </td>
+  expression mVenus. A:Cells detected in bright field picture B: Detection of
-      <td
+  mVenus expression can be observed.</span></p>
- 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;"
+  </td>
- valign="top" width="307">
+ </tr>
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">D</p>
-      <p class="MsoNormal"
- style="margin-bottom: 0.0001pt; text-align: center; text-indent: 0cm; line-height: normal;"
- align="center"><img
- src="Freiburg10_Modularization_GOI_02_files/image026.jpg"
- alt="Description: Description: https://static.igem.org/mediawiki/2010/4/40/2010-7-8_plate_1_A_2_solo_cell.jpg"
- height="194" width="257"></p>
-      </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">Figure </a>6: <span
- style="font-weight: normal;">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">&nbsp;</p>
-<h4 style="margin-left: 0cm; text-indent: 0cm;"><span
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
- lang="DE">Analysis </span>of
-Target Cells by Flow Cytometry demonstrates GOI Expression</h4>
+<h4 style='margin-left:0cm;text-indent:0cm'><a name="_Toc275796906"><span
-<p class="MsoNormal">Characterizing the function of the
+lang=EN-US>Analysis of Target Cells by Flow Cytometry demonstrates GOI
-hGH terminator, the <i>beta-globin</i>
+Expression</span></a></h4>
-intron and the complete plasmid, several approaches were conducted
-followed by analysis
+<p class=MsoNormal><span lang=EN-US>Characterizing the function of the hGH
-via flow cytometry. </p>
+terminator, the <i>beta-globin</i> intron and the complete plasmid, several
-<h5>Influence of hGH terminator BioBrick on GOI Expression</h5>
+approaches were conducted followed by analysis via flow cytometry. </span></p>
-<p class="MsoNormal">The iGEM team Freiburg provides the
-hGH plolyadenylation
+<h5><a name="_Toc275796907"><span lang=EN-US>Influence of hGH terminator
-sequence within the ‘Virus Construction Kit’ due to the fact that
+BioBrick on GOI Expression</span></a></h5>
-almost every
-eukaryotic mRNA is processed at their 3´ and 5´end except for histone
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg provides the hGH
-mRNAs
+plolyadenylation sequence within the ‘Virus Construction Kit’ due to the fact
-(Millevoi et al. 2006). Pre-mRNAs contain two canonical conserved
+that almost every eukaryotic mRNA is processed at their 3´ and 5´end except for
-sequences.
+histone mRNAs </span><span
-First, the polyadenylation signal “AATAAA” which is recognized by the
+lang=EN-US>(Millevoi et al. 2006)</span><span lang=EN-US>. Pre-mRNAs contain
-multiprotein complex and second the GT-rich region (downstream sequence
+two canonical conserved sequences. First, the polyadenylation signal “AATAAA”
-element, DSE) which is located 30 nucleotides downstream of the
+which is recognized by the multiprotein complex and second the GT-rich region
-cleavage site.
+(downstream sequence element, DSE) which is located 30 nucleotides downstream
-The assembled 3´end-processing machinery cleaves the mRNA transcript
+of the cleavage site. The assembled 3´end-processing machinery cleaves the mRNA
-immediately after a CA-nucleotide therefore defining the cleavage site
+transcript immediately after a CA-nucleotide therefore defining the cleavage
-(Danckwardt et al. 2008)<span
+site </span><span
- style="font-size: 12pt; line-height: 200%;">. </span>Recombinant
+lang=EN-US>(Danckwardt et al. 2008)</span><span lang=EN-US style='font-size:
-vectorplasmids were engineered containing the inverted terminal repeats
+.0pt;line-height:200%'>. </span><span lang=EN-US>Recombinant vectorplasmids
-(ITRs),
+were engineered containing the inverted terminal repeats (ITRs), a strong
-a strong eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as
+eukaryotic promoter (CMV promoter: BBa_K404102) and mVenus as gene of interest
-gene of
+with and without the hGH terminator signal. Transduction of HT1080 cells with constant
-interest with and without the hGH terminator signal. Transduction of
+volume of viral particles containing the vectorplasmids and measuring mVenus
-HT1080
+expression 24-hours post infection by flow cytometry demonstrated that
-cells with constant volume of viral particles containing the
+transgene expression of the constructs lacking the hGH termination signal is
-vectorplasmids and
+significantly reduced as shown in </span><span
-measuring mVenus expression 24-hours post infection by flow cytometry
+lang=EN-US>Figure 7</span><span lang=EN-US> and </span><span
-demonstrated that transgene expression of the constructs lacking the
+lang=EN-US>Figure 8</span><span lang=EN-US> confirming the expected results
-hGH
+that hGH is essential for mRNA processing. The iGEM team Freiburg_Bioware 2010
-termination signal is significantly reduced as shown in Figure 7 and
+therefore suggests using the provided hGH termination signal within the Virus
-Figure 8
+Construction Kit for optimal gene expression.</span></p>
-confirming the expected results that hGH is essential for mRNA
-processing. The
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-iGEM team Freiburg_Bioware 2010 therefore suggests using the provided
+  style='border-collapse:collapse;border:none'>
-hGH
+  <tr>
-termination signal within the Virus Construction Kit for optimal gene
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-expression.</p>
+  padding:0cm 5.4pt 0cm 5.4pt'>
-<table class="MsoNormalTable"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-  style="border-collapse: collapse;" border="0"
+cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid lacking hGH
-  cellpadding="0" cellspacing="0">
+  termination signal</span></b></p>
-  <tbody>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-    <tr>
+cm;line-height:normal'><img width=629 height=408 id="Grafik 30"
-      <td
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image027.gif"></p>
- style="border: 1pt solid windowtext; 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>Vectorplasmid
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-lacking hGH termination signal</b></p>
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt'>
-      <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;"><img
+cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid containing hGH terminator
- src="Freiburg10_Modularization_GOI_02_files/image027.gif"
+  signal</span></b></p>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image027.gif"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
- height="408" width="629"></p>
+cm;line-height:normal;page-break-after:avoid'><img width=635 height=410
-      </td>
+  id="Grafik 2049"
-    </tr>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image028.gif"></p>
-    <tr>
+  </td>
-      <td
+ </tr>
- 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;"
+ <tr>
- valign="top" width="641">
+  <td width=641 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;"><b>Vectorplasmid
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-containing hGH terminator signal</b></p>
+cm;line-height:normal'><a name="_Ref275784539"><b><span lang=EN-US>Figure </span></b></a><b><span lang=EN-US>7</span></b><b><span lang=EN-US>:</span></b><span lang=EN-US> <b>Flow
-      <p class="MsoNormal"
+  cytometry analysis of vectorplasmids with and without hGH terminator.</b> </span></p>
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
- src="Freiburg10_Modularization_GOI_02_files/image028.gif"
+cm;line-height:normal'><b><span lang=EN-US>A</span></b><span lang=EN-US>:
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image028.gif"
+  Gating non transduced cells (control); subcellular debris and clumps can be
- height="410" width="636"></p>
+  distinguished from single cells by size, estimated forward scatter (FS Lin)
-      </td>
+  and granularity, estimated side scatter (SS Lin) <b>B</b>: Non transduced
-    </tr>
+  cells applied against mVenus (Analytical gate was set such that 1% or fewer
-    <tr>
+  of negative control cells fell within the positive region (R5). <b>C</b>:
-      <td
+  Gating transduced cells (R2 </span><span lang=EN-US style='font-family:"Cambria Math","serif"'>&#8793;</span><span
- 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;"
+  lang=EN-US>R14) (used plasmids for transfection: GOI: <b>pSB1C3_lITR_CMV_beta-globin
- valign="top" width="641">
+  intron_mVenus_rITR (BBa_K404127)</b>, pHelper, pRC). <b>D</b>: Transduced
-      <p class="MsoNormal"
+  cells plotted against mVenus, R10 comprises transduced cells by detecting
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><a
+  mVenus expression. <b>E</b>: Overlay of non-transduced (red) and transduced
- name="_Ref275784539"><b>Figure </b></a><b>7:</b>
+  (green) cells applied against mVenus.<b>F</b>: Gating non-transduced cells
-      <b>Flow cytometry analysis of vectorplasmids with and
+  (control) <b>G</b>: Non-transduced cells applied against mVenus. <b>H</b>:
-without hGH terminator.</b> </p>
+  Gating transduced cells (R2 </span><span lang=EN-US style='font-family:"Cambria Math","serif"'>&#8793;</span><span
-      <p class="MsoNormal"
+  lang=EN-US>R14) (used plasmids for transfection: GOI: reassembled <b>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>A</b>:
+  (BBa_K404119)</b>, pHelper, pRC). <b>I</b>: Transduced cells applied against
-Gating non transduced cells (control); subcellular debris and clumps
+  mVenus, R10 comprised transduced cells, by detecting mVenus expression. <b>J</b>:
-can be distinguished from single cells by size, estimated forward
+  Overlay of non-transduced (red) and transduced (green) cells applied against
-scatter (FS Lin) and granularity, estimated side scatter (SS Lin) <b>B</b>:
+  mVenus.</span></p>
-Non transduced cells applied against mVenus (Analytical gate was set
+  </td>
-such that 1% or fewer of negative control cells fell within the
+ </tr>
-positive region (R5). <b>C</b>: Gating transduced cells
-(R2 <span style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)
-(used plasmids for transfection: GOI: <b>pSB1C3_lITR_CMV_beta-globin
-intron_mVenus_rITR (BBa_K404127)</b>, pHelper, pRC). <b>D</b>:
-Transduced cells plotted against mVenus, R10 comprises transduced cells
-by detecting mVenus expression. <b>E</b>: Overlay of
-non-transduced (red) and transduced (green) cells applied against
-mVenus.<b>F</b>: Gating non-transduced cells (control) <b>G</b>:
-Non-transduced cells applied against mVenus. <b>H</b>:
-Gating transduced cells (R2 <span
- style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;;">≙</span>R14)
-(used plasmids for transfection: GOI: reassembled <b>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-(BBa_K404119)</b>, pHelper, pRC). <b>I</b>:
-Transduced cells applied against mVenus, R10 comprised transduced
-cells, by detecting mVenus expression. <b>J</b>: Overlay
-of non-transduced (red) and transduced (green) cells applied against
-mVenus.</p>
-      </td>
-    </tr>
-  </tbody>
 </table>
-<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
-<table class="MsoNormalTable"
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-  style="border-collapse: collapse;" border="0"
-  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;">&nbsp;</p>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-      <p class="MsoNormal"
+cm;line-height:normal;page-break-after:avoid'><span lang=EN-US>                          </span><img
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;">&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
+  width=473 height=355 id="Diagramm 3"
-      <img src="Freiburg10_Modularization_GOI_02_files/image029.gif"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image029.gif"></p>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image029.gif"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784545"><span
- height="355" width="473"></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>8</span><span lang=EN-US>: Flow
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
+  cytometry analysis of vectorplasmids with and without hGH terminator.</span><span
- name="_Ref275784545">Figure </a>8: Flow cytometry
+  lang=EN-US style='font-weight:normal'> YFP expression of viral genomes was
-analysis of vectorplasmids with and without hGH terminator.<span
+  determined by flow cytomery after 24-hour post infection. Results demonstrate
- style="font-weight: normal;"> YFP expression of viral
+  that mVenus expression of vectorplasmids lacking the hGH terminator is
-genomes was determined by flow cytomery after 24-hour post infection.
+  reduced significantly proving that the polyadenylation signal is essential
-Results demonstrate that mVenus expression of vectorplasmids lacking
+  for viral gene expression using recombinant viral vectors engineered by using
-the hGH terminator is reduced significantly proving that the
+  components of the Virus Construction Kit.</span></p>
-polyadenylation signal is essential for viral gene expression using
+  </td>
-recombinant viral vectors engineered by using components of the Virus
+ </tr>
-Construction Kit.</span></p>
-      </td>
-    </tr>
-  </tbody>
 </table>
-<p class="MsoCaption">&nbsp;</p>
-<h5>Influence of <i>Beta-globin</i> intron Biobrick
+<p class=MsoCaption><span lang=EN-US>&nbsp;</span></p>
-on GOI Expression</h5>
-<p class="MsoNormal">Providing an element assumed to be an
+<h5><a name="_Toc275796908"><span lang=EN-US>Influence of <i>Beta-globin</i>
-enhancer of transgene
+intron Biobrick on GOI Expression</span></a></h5>
-expression (Nott et al. 2003), the iGEM team Freiburg tested a
-beta-globin
+<p class=MsoNormal><span lang=EN-US>Providing an element assumed to be an
-intron derived from the human <i>beta globin</i> gene
+enhancer of transgene expression </span><span
-which can be fused
+lang=EN-US>(Nott et al. 2003)</span><span lang=EN-US>, the iGEM team Freiburg tested
-upstream of the desired gene of interest. The beta-globin intron
+a beta-globin intron derived from the human <i>beta globin</i> gene which can
-BioBrick
+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
+consists of a partial chimeric CMV promoter followed by the intron II of the <i>beta-globin</i>
-of the <i>beta-globin</i>
 gene. The 3´end of the intron is fused to the first 25 bases of human <i>beta
-globin</i> gene exon 3. The <i>beta globin</i>
+globin</i> gene exon 3. The <i>beta globin</i> intron BioBrick is assumed to
-intron BioBrick is assumed to
+enhance eukaryotic gene expression </span><span
-enhance eukaryotic gene expression (Nott et al. 2003). Analysis was
+lang=EN-US>(Nott et al. 2003)</span><span lang=EN-US>. Analysis was conducted
-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
-as described for the hGH terminator experiment (see above). As shown in
+lang=EN-US>Figure 10</span><span lang=EN-US> the vectorplasmid missing the <i>beta-globin</i>
-Figure
+intron showed a negligible difference in mVenus expression compared to viral
-and Figure 10 the vectorplasmid missing the <i>beta-globin</i>
+genomes containing the <i>beta-globin</i> intron. Considering these results and
-intron showed
+taking into account that a constant volume of viral particles has been used for
-a negligible difference in mVenus expression compared to viral genomes
+transduction, the difference between the construct containing and lacking the
-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 (Dong et al. 1996), the
+decreases with increasing sizes of the insert </span><span
-iGEM team
+lang=EN-US>(Dong et al. 1996)</span><span lang=EN-US>, the iGEM team
-Freiburg_Bioware suggests using the <i>beta-globin </i>intron
+Freiburg_Bioware suggests using the <i>beta-globin </i>intron in dependence on
-in dependence on
+the size of your transgene.</span></p>
-the size of your transgene.</p>
-<table class="MsoNormalTable"
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 width=654
-  style="width: 490.75pt; border-collapse: collapse;" border="0"
+  style='width:490.75pt;border-collapse:collapse;border:none'>
- 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>Vectorplasmid
+cm;line-height:normal'><img width=640 height=412 id="Grafik 55"
-lacking <i>beta-globin</i> intron</b></p>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image030.gif"></p>
-      <p class="MsoNormal"
+  </td>
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><img
+ </tr>
- src="Freiburg10_Modularization_GOI_02_files/image030.gif"
+ <tr style='height:106.5pt'>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image030.gif"
+  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
- height="412" width="640"></p>
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:106.5pt'>
-      </td>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-    </tr>
+cm;line-height:normal'><b><span lang=EN-US>Vectorplasmid containing <i>beta-globin</i>
-    <tr style="height: 106.5pt;">
+  intron</span></b></p>
-      <td
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
- 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;"
+cm;line-height:normal;page-break-after:avoid'><img width=635 height=410
- valign="top" width="654">
+  id="Grafik 63"
-      <p class="MsoNormal"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image031.gif"></p>
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b>Vectorplasmid
+  </td>
-containing <i>beta-globin</i> intron</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;
- src="Freiburg10_Modularization_GOI_02_files/image031.gif"
+  border-top:none;padding:0cm 5.4pt 0cm 5.4pt;height:106.5pt'>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image031.gif"
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784803"><span
- height="410" width="636"></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>9</span><span lang=EN-US>: Flow
-      </td>
+  cytometry analysis of vectorplasmids with and without <i>beta-globin</i>
-    </tr>
+  intron.  A</span><span lang=EN-US style='font-weight:normal'>: Gating non
-    <tr style="height: 106.5pt;">
+  transduced cells (control); subcellular debris and clumps can be
-      <td
+  distinguished from single cells by size, estimated forward scatter (FS Lin)
- 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;"
+  and granularity, estimated side scatter (SS Lin) </span><span lang=EN-US>B</span><span
- valign="top" width="654">
+  lang=EN-US style='font-weight:normal'>: Non transduced cells applied against
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
+  mVenus (Analytical gate was set such that 1% or fewer of negative control
- name="_Ref275784803">Figure </a>9: Flow cytometry
+  cells fell within the positive region (R5). </span><span lang=EN-US>C</span><span
-analysis of vectorplasmids with and without <i>beta-globin</i>
+  lang=EN-US style='font-weight:normal'>: Gating transduced cells (R2 </span><span
-intron.&nbsp; A<span style="font-weight: normal;">:
+  lang=EN-US style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
-Gating non transduced cells (control); subcellular debris and clumps
+  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
-can be distinguished from single cells by size, estimated forward
+  GOI: </span><span lang=EN-US>pSB1C3_lITR_CMV_mVenus_hGH_rITR (BBa_K404128)</span><span
-scatter (FS Lin) and granularity, estimated side scatter (SS Lin) </span>B<span
+  lang=EN-US style='font-weight:normal'>, pHelper, pRC). </span><span
- style="font-weight: normal;">: Non transduced cells applied
+  lang=EN-US>D</span><span lang=EN-US style='font-weight:normal'>: Transduced
-against mVenus (Analytical gate was set such that 1% or fewer of
+  cells plotted against mVenus, R10 comprised transduced cells, by detecting
-negative control cells fell within the positive region (R5). </span>C<span
+  mVenus expression </span><span lang=EN-US>E</span><span lang=EN-US
- style="font-weight: normal;">: Gating transduced cells (R2 </span><span
+  style='font-weight:normal'>: Overlay of non-transduced (red) and transduced
- style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
+  (green) cells applied against mVenus </span><span lang=EN-US>F</span><span
- style="font-weight: normal;">R14) (used plasmids for
+  lang=EN-US style='font-weight:normal'>: Gating non-transduced cells (control).
-transfection: GOI: </span>pSB1C3_lITR_CMV_mVenus_hGH_rITR
+  </span><span lang=EN-US>G</span><span lang=EN-US style='font-weight:normal'>:
-(BBa_K404128)<span style="font-weight: normal;">, pHelper,
+  Non-transduced cells applied against mVenus (R5).</span><span lang=EN-US>H</span><span
-pRC). </span>D<span style="font-weight: normal;">:
+  lang=EN-US style='font-weight:normal'>: Gating transduced cells (R2 </span><span
-Transduced cells plotted against mVenus, R10 comprised transduced
+  lang=EN-US style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
-cells, by detecting mVenus expression </span>E<span
+  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
- style="font-weight: normal;">: Overlay of non-transduced
+  GOI: reassembled </span><span lang=EN-US>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-(red) and transduced (green) cells applied against mVenus </span>F<span
+  (BBa_K404119)</span><span lang=EN-US style='font-weight:normal'>, pHelper, pRC).
- style="font-weight: normal;">: Gating non-transduced cells
+  </span><span lang=EN-US>I</span><span lang=EN-US style='font-weight:normal'>:
-(control). </span>G<span style="font-weight: normal;">:
+  Transduced cells applied against mVenus, R10 comprised transduced cells, by
-Non-transduced cells applied against mVenus (R5).</span>H<span
+  detecting mVenus expression. </span><span lang=EN-US>J</span><span
- style="font-weight: normal;">: Gating transduced cells (R2 </span><span
+  lang=EN-US style='font-weight:normal'>: Overlay of non-transduced (red) and
- style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
+  transduced (green) cells applied against mVenus.</span></p>
- style="font-weight: normal;">R14) (used plasmids for
+  </td>
-transfection: GOI: reassembled </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+ </tr>
-(BBa_K404119)<span style="font-weight: normal;">, pHelper,
-pRC). </span>I<span style="font-weight: normal;">:
-Transduced cells applied against mVenus, R10 comprised transduced
-cells, by detecting mVenus expression. </span>J<span
- style="font-weight: normal;">: Overlay of non-transduced
-(red) and transduced (green) cells applied against mVenus.</span></p>
-      </td>
-    </tr>
-  </tbody>
 </table>
-<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
-<table class="MsoNormalTable"
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-  style="width: 490.75pt; border-collapse: collapse;" border="0"
- cellpadding="0" cellspacing="0" width="654">
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0 width=654
-  <tbody>
+  style='width:490.75pt;border-collapse:collapse;border:none'>
-    <tr style="height: 90.2pt;">
+ <tr style='height:90.2pt'>
-      <td
+  <td width=654 valign=top style='width:490.75pt;border:solid windowtext 1.0pt;
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 490.75pt; height: 90.2pt;"
+  padding:0cm 5.4pt 0cm 5.4pt;height:90.2pt'>
- valign="top" width="654">
+  <p class=MsoNormal align=left style='margin-bottom:0cm;margin-bottom:.0001pt;
-      <p class="MsoNormal"
+  text-align:left;text-indent:0cm;line-height:normal'><span lang=EN-US>&nbsp;</span></p>
- style="margin-bottom: 0.0001pt; text-align: left; text-indent: 0cm; line-height: normal;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- align="left">&nbsp;</p>
+  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%20B&amp;K_3-Dateien/image032.gif"></p>
- align="center"><img
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784805"><span
- src="Freiburg10_Modularization_GOI_02_files/image032.gif"
+  lang=EN-US>Figure </span></a><span lang=EN-US>10</span><span lang=EN-US>:
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image032.gif"
+  Flow cytometry analysis of vectorplasmids with and without <i>beta-globin</i>
- height="332" width="450"></p>
+  intron.</span><span lang=EN-US style='font-weight:normal'> 48-hours post
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
+  transfection, viral particles were harvested by freeze-thaw lysis and
- name="_Ref275784805">Figure </a>10: Flow cytometry
+  centrifugation followed by HT1080 transduction. YFP expression of vectorplasmids
-analysis of vectorplasmids with and without <i>beta-globin</i>
+  was determined by flow cytometry 24-hours post infection. The vectorplasmid
-intron.<span style="font-weight: normal;"> 48-hours post
+  missing the beta-globin intron showed a negligible difference in mVenus
-transfection, viral particles were harvested by freeze-thaw lysis and
+  expression compared to viral plasmid containing the beta-globin intron.</span></p>
-centrifugation followed by HT1080 transduction. YFP expression of
+  </td>
-vectorplasmids was determined by flow cytometry 24-hours post
+ </tr>
-infection. The vectorplasmid 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;">&nbsp;</p>
-<h5>Functionality of the Full Assembled Vectorplasmid
+<p class=MsoNormal style='text-indent:0cm'><span lang=EN-US>&nbsp;</span></p>
-Demonstrated by GOI
-Expression </h5>
+<h5><a name="_Toc275796909"><span lang=EN-US>Functionality of the Full
-<p class="MsoNormal">After assembly of plasmids containing
+Assembled Vectorplasmid Demonstrated by GOI Expression</span></a><span
-all required elements
+lang=EN-US> </span></h5>
-(see Figure 1), functionality was tested in cell culture. AAV-293 cells
-stably
+<p class=MsoNormal><span lang=EN-US>After assembly of plasmids containing all required
-expressing E1A and E1B proteins were transfected with three plasmids
+elements (see </span><span lang=EN-US>Figure 1</span><span lang=EN-US>),
-&nbsp;(pHelper, pRC, pGOI). Virus particles were harvested 72-hours
+functionality was tested in cell culture. AAV-293 cells stably expressing E1A
-post-transfection and the tumor cell line HT1080 was transduced with
+and E1B proteins were transfected with three plasmids  (pHelper, pRC, pGOI).
-the
+Virus particles were harvested 72-hours post-transfection and the tumor cell
-recombinant viral vectors encapsidating the gene of interest mVenus
+line HT1080 was transduced with the recombinant viral vectors encapsidating the
-(BBa_I757008).</p>
+gene of interest mVenus (BBa_I757008).</span></p>
-<p class="MsoNormal">The iGEM team Freiburg_Bioware 2010
-compared the
+<p class=MsoNormal><span lang=EN-US>The iGEM team Freiburg_Bioware 2010
-standard-plasmid containing a subcloned mVenus (pAAV_mVenus, derived
+compared the standard-plasmid containing a subcloned mVenus (pAAV_mVenus,
-from the
+derived from the Stratagene system) with the assembled plasmid pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-Stratagene system) with the assembled plasmid
+(pSB1C3_mVenus: BBa_K404119). Fluorescence expression data obtained by flow
-pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR (pSB1C3_mVenus:
+cytometry analysis are shown in </span><span lang=EN-US>Figure 11</span><span
-BBa_K404119).
+lang=EN-US> and </span><span lang=EN-US>Figure 12</span><span lang=EN-US>.
-Fluorescence expression data obtained by flow cytometry analysis are
+Comparing mVenus expression of the standard plasmid and the modified, assembled
-shown in
+plasmid reveals that biological functionality of the reassembled plasmid was
-Figure 11 and Figure 12. Comparing mVenus expression of the standard
+confirmed. </span></p>
-plasmid
-and the modified, assembled plasmid reveals that biological
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-functionality of
+  style='border-collapse:collapse;border:none'>
-the reassembled plasmid was confirmed. </p>
+ <tr>
-<table class="MsoNormalTable"
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-  style="border-collapse: collapse;" border="0"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- cellpadding="0" cellspacing="0">
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-  <tbody>
+cm;line-height:normal'><b>pSB1C3_mVenus (BBa_K404119)</b></p>
-    <tr>
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
-      <td
+cm;line-height:normal'><img width=634 height=409 id="Grafik 2068"
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image033.gif"></p>
- valign="top" width="641">
+  </td>
-      <p class="MsoNormal"
+ </tr>
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;"><b><span
+ <tr>
- lang="DE">pSB1C3_mVenus (BBa_K404119)</span></b></p>
+  <td width=641 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;"><img
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
- src="Freiburg10_Modularization_GOI_02_files/image033.gif"
+cm;line-height:normal'><b><span lang=EN-US>pAAV_mVenus (Stratagene)</span></b></p>
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image033.gif"
+  <p class=MsoNormal style='margin-bottom:0cm;margin-bottom:.0001pt;text-indent:
- height="409" width="634"></p>
+cm;line-height:normal;page-break-after:avoid'><img width=630 height=407
-      </td>
+  id="Grafik 89"
-    </tr>
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image034.gif"></p>
-    <tr>
+  </td>
-      <td
+ </tr>
- 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;"
+ <tr>
- valign="top" width="641">
+  <td width=641 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;"><b>pAAV_mVenus
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784576"><span
-(Stratagene)</b></p>
+  lang=EN-US>Figure </span></a><span lang=EN-US>11</span><span lang=EN-US>: Flow
-      <p class="MsoNormal"
+  cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared to
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal; page-break-after: avoid;"><img
+  standard plasmid provided by Stratagene. A</span><span lang=EN-US
- src="Freiburg10_Modularization_GOI_02_files/image034.gif"
+  style='font-weight:normal'>: Gating non transduced cells (control);
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image034.gif"
+  subcellular debris and clumps can be distinguished from single cells by size,
- height="406" width="630"></p>
+  estimated forward scatter (FS Lin) and granularity, estimated side scatter
-      </td>
+  (SS Lin) B: Non transduced cells plotted against mVenus (Analytical gate was
-    </tr>
+  set such that 1% or fewer of negative control cells fell within the positive
-    <tr>
+  region (R5).C: Gating transduced cells (R2 </span><span lang=EN-US
-      <td
+  style='font-family:"Cambria Math","serif";font-weight:normal'>&#8793;</span><span
- 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;"
+  lang=EN-US style='font-weight:normal'>R14) (used plasmids for transfection:
- valign="top" width="641">
+  pGOI: </span><span lang=EN-US>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
+  (pSB1C3_mVenus: </span><span lang=EN-US>BBa_K404119</span><span lang=EN-US
- name="_Ref275784576">Figure </a>11: Flow cytometry
+  style='color:#00B050;font-weight:normal'>)</span><span lang=EN-US
-analysis of reassembled vectorplasmid (BBa_K404119) compared to
+  style='font-weight:normal'>, pHelper, pRC. </span><span lang=EN-US>D</span><span
-standard plasmid provided by Stratagene. A<span
+  lang=EN-US style='font-weight:normal'>: Transduced cells plotted against
- style="font-weight: normal;">: Gating non transduced cells
+  mVenus, R10 comprised transduced cells, by detecting mVenus expression. </span><span
-(control); subcellular debris and clumps can be distinguished from
+  lang=EN-US>E</span><span lang=EN-US style='font-weight:normal'>: Overlay of
-single cells by size, estimated forward scatter (FS Lin) and
+  non-transduced (red) and transduced (green). </span><span lang=EN-US>F</span><span
-granularity, estimated side scatter (SS Lin) B: Non transduced cells
+  lang=EN-US style='font-weight:normal'>: Gating non transduced cells
-plotted against mVenus (Analytical gate was set such that 1% or fewer
+  (control). </span><span lang=EN-US>G</span><span lang=EN-US style='font-weight:
-of negative control cells fell within the positive region (R5).C:
+  normal'>: Non-transduced cells plotted against mVenus (R5). </span><span
-Gating transduced cells (R2 </span><span
+  lang=EN-US>H</span><span lang=EN-US style='font-weight:normal'>: Gating
- style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
+  transduced cells (R14 </span><span lang=EN-US style='font-family:"Cambria Math","serif";
- style="font-weight: normal;">R14) (used plasmids for
+  font-weight:normal'>&#8793;</span><span lang=EN-US style='font-weight:normal'>R2)
-transfection: pGOI: </span>pSB1C3_lITR_CMV_beta-globin_mVenus_hGH_rITR
+  (used plasmids for transfection: pGOI: pAAV_mVenus, pHelper). </span><span
-(pSB1C3_mVenus: BBa_K404119<span
+  lang=EN-US>I</span><span lang=EN-US style='font-weight:normal'>: Transduced
- style="color: rgb(0, 176, 80); font-weight: normal;">)</span><span
+  cells plotted against mVenus, R10 comprised transduced cells, by detecting
- style="font-weight: normal;">, pHelper, pRC. </span>D<span
+  mVenus expression.</span><span lang=EN-US> J</span><span lang=EN-US
- style="font-weight: normal;">: Transduced cells plotted
+  style='font-weight:normal'>: Overlay of non-transduced (red) and transduced
-against mVenus, R10 comprised transduced cells, by detecting mVenus
+  (green) cells plotted against mVenus expression. </span></p>
-expression. </span>E<span style="font-weight: normal;">:
+  </td>
-Overlay of non-transduced (red) and transduced (green). </span>F<span
+ </tr>
- style="font-weight: normal;">: Gating non transduced cells
-(control). </span>G<span style="font-weight: normal;">:
-Non-transduced cells plotted against mVenus (R5). </span>H<span
- style="font-weight: normal;">: Gating transduced cells (R14 </span><span
- style="font-family: &quot;Cambria Math&quot;,&quot;serif&quot;; font-weight: normal;">≙</span><span
- style="font-weight: normal;">R2) (used plasmids for
-transfection: pGOI: pAAV_mVenus, pHelper). </span>I<span
- style="font-weight: normal;">: Transduced cells plotted
-against mVenus, R10 comprised transduced cells, by detecting mVenus
-expression.</span> J<span style="font-weight: normal;">:
-Overlay of non-transduced (red) and transduced (green) cells plotted
-against mVenus expression. </span></p>
-      </td>
-    </tr>
-  </tbody>
 </table>
-<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
-<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-<p class="MsoNormal"><span lang="DE">&nbsp;</span></p>
-<table class="MsoNormalTable"
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-  style="border-collapse: collapse;" border="0"
- cellpadding="0" cellspacing="0">
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
-  <tbody>
-    <tr>
+<table class=MsoTableGrid border=1 cellspacing=0 cellpadding=0
-      <td
+  style='border-collapse:collapse;border:none'>
- style="border: 1pt solid windowtext; padding: 0cm 5.4pt; width: 481.1pt;"
+ <tr>
- valign="top" width="641">
+  <td width=641 valign=top style='width:481.1pt;border:solid windowtext 1.0pt;
-      <p class="MsoNormal"
+  padding:0cm 5.4pt 0cm 5.4pt'>
- style="margin-bottom: 0.0001pt; text-indent: 0cm; line-height: normal;">&nbsp;</p>
+  <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-align: center; text-indent: 0cm; line-height: normal; page-break-after: avoid;"
+  <p class=MsoNormal align=center style='margin-bottom:0cm;margin-bottom:.0001pt;
- align="center"><img
+  text-align:center;text-indent:0cm;line-height:normal;page-break-after:avoid'><img
- src="Freiburg10_Modularization_GOI_02_files/image035.gif"
+  width=541 height=396 id="Diagramm 90"
- alt="Description: D:\user\kristian\iGEM\iGEM2010\wiki_igem\Freiburg10_Modularization_GOI_02_files\image035.gif"
+  src="Freiburg10_Modularization_GOI%20B&amp;K_3-Dateien/image035.gif"></p>
- height="396" width="541"></p>
+  <p class=MsoCaption style='text-indent:0cm'><a name="_Ref275784852"><span
-      <p class="MsoCaption" style="text-indent: 0cm;"><a
+  lang=EN-US>Figure </span></a><span lang=EN-US>12</span><span lang=EN-US>:
- name="_Ref275784852">Figure </a>12: Flow cytometry
+  Flow cytometry analysis of reassembled vectorplasmid (BBa_K404119) compared
-analysis of reassembled vectorplasmid (BBa_K404119) compared to
+  to standard plasmid provided by Stratagene. </span><span lang=EN-US
-standard plasmid provided by Stratagene. <span
+  style='font-weight:normal'>Fluorescence of the standard plasmid pAAV_mVenus (Stratagene)
- style="font-weight: normal;">Fluorescence of the standard
+  and the recombinant pSB1C3_mVenus (BBa_K404119) construct was measured. As
-plasmid pAAV_mVenus (Stratagene) and the recombinant pSB1C3_mVenus
+  demonstrated mVenus expression is enhanced in the assembled plasmid
-(BBa_K404119) construct was measured. As demonstrated mVenus expression
+  (pSB1C3_mVenus) compared to the standard pAAV_mVenus construct.</span></p>
-is enhanced in the assembled plasmid (pSB1C3_mVenus) compared to the
+  </td>
-standard pAAV_mVenus construct.</span></p>
+ </tr>
-      </td>
-    </tr>
-  </tbody>
 </table>
-<p class="MsoNormal">&nbsp;</p>
-<p style="margin-left: 24pt; text-indent: -24pt;"><span
+<p class=MsoNormal><span lang=EN-US>&nbsp;</span></p>
- style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
- lang="DE">Danckwardt, S.,
+<p style='margin-left:24.0pt;text-indent:-24.0pt'><span
-Hentze, M.W. &amp; Kulozik, A.E., 2008. 3’ end mRNA processing:
+lang=EN-US style='font-size:10.0pt;font-family:"Calibri","sans-serif"'>Danckwardt,
-molecular
+S., Hentze, M.W. &amp; Kulozik, A.E., 2008. 3’ end mRNA processing: molecular
-mechanisms and implications for health and disease. <i>The EMBO
+mechanisms and implications for health and disease. <i>The EMBO journal</i>,
-journal</i>,
+(3), pp.482-98. Available at: http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
-(3), pp.482-98. Available at:
-http://www.ncbi.nlm.nih.gov/pubmed/18256699.</span></p>
+<p style='margin-left:24.0pt;text-indent:-24.0pt'><span lang=EN-US
-<p style="margin-left: 24pt; text-indent: -24pt;"><span
+style='font-size:10.0pt;font-family:"Calibri","sans-serif"'>Dong, J.Y., Fan,
- style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+P.D. &amp; Frizzell, R.A., 1996. Quantitative analysis of the packaging
- lang="DE">Dong, J.Y., Fan,
+capacity of recombinant adeno-associated virus. <i>Human gene therapy</i>,
-P.D. &amp; Frizzell, R.A., 1996. Quantitative analysis of the
+(17), pp.2101-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
-packaging capacity
-of recombinant adeno-associated virus. <i>Human gene therapy</i>,
+<p style='margin-left:24.0pt;text-indent:-24.0pt'><span lang=EN-US
-(17),
+style='font-size:10.0pt;font-family:"Calibri","sans-serif"'>Millevoi, S. et
-pp.2101-12. Available at: http://www.ncbi.nlm.nih.gov/pubmed/8934224.</span></p>
+al., 2006. An interaction between U2AF 65 and CF I(m) links the splicing and 3’
-<p style="margin-left: 24pt; text-indent: -24pt;"><span
+end processing machineries. <i>The EMBO journal</i>, 25(20), pp.4854-64.
- style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
- lang="DE">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>,
-(20), pp.4854-64.
 Available at: http://www.ncbi.nlm.nih.gov/pubmed/17024186.</span></p>
-<p style="margin-left: 24pt; text-indent: -24pt;"><span
- style="font-size: 10pt; font-family: &quot;Calibri&quot;,&quot;sans-serif&quot;;"
+<p style='margin-left:24.0pt;text-indent:-24.0pt'><span lang=EN-US
- lang="DE">Nott, A., Meislin,
+style='font-size:10.0pt;font-family:"Calibri","sans-serif"'>Nott, A., Meislin,
-S.H. &amp; Moore, M.J., 2003. A quantitative analysis of intron
+S.H. &amp; Moore, M.J., 2003. A quantitative analysis of intron effects on
-effects on
+mammalian gene expression. <i>RNA (New York, N.Y.)</i>, 9(5), pp.607-17.
-mammalian gene expression. <i>RNA (New York, N.Y.)</i>,
-(5), pp.607-17.
 Available at: http://www.ncbi.nlm.nih.gov/pubmed/12702819.</span></p>
-<p style="margin-left: 24pt; text-indent: -24pt;">&nbsp;</p>
+<p style='margin-left:24.0pt;text-indent:-24.0pt'><span lang=EN-US>&nbsp;</span></p>
 </html>