Team:Nanjing China Bio/Safety2

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                         <li><a href="/Team:Nanjing_China_Bio/results">Parts Submitted</a></li>
                         <li><a href="/Team:Nanjing_China_Bio/results">Parts Submitted</a></li>
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                             <li><a href="/Team:Nanjing_China_Bio/Safety2" class="hover">Safety</a></li>   
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                             <li><a href="/Team:Nanjing_China_Bio/Safety2" class="hover">Data</a></li>   
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1.Would any of your project ideas raise safety issues in terms of:
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      To confirm the distribution of VNP20009 in mouse tissues and to verify the tumor-specific gene induction, we detected GFP expression in tumor, liver, and spleen by fluorescence microscopy. We found the VNPpLacZ-GFP (with constitutive promoter) could induce expression not only in necrotic tumor but also in spleen and liver tissue. However, VNPpNirBeGFP (with tumor specific promoter) could only be induced in hypoxic and necrotic tumor tissue. <br/>
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Researcher safety, Public safety, or Environment safety<br>
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      <br/><div style="width:558px; height:462px; margin:0 auto;"><img src="https://static.igem.org/mediawiki/igem.org/a/af/122.jpg"></div>
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  Based on our experimental design, all facilities in the laboratory are performed in Biosafety Level 2 and the materials we use are E.coli Top 10, Salmonella typhimurium VNP, Melanoma B16F10 and the laboratory mice. Since the Salmonella typhimurium VNP is infectious, we take some measures to ensure the health of the team when we conduct the experiments with them. During all the experiments, all the instructions are posted and the workers are asked to wear the nitrile gloves, lab coats and masks, moreover, a room is separated for the experiments and the bacterial is kept in a special place. We also clean the experiment table once a day. For the researcher safety, our team is trained by all the safety regulations before we start the experiments. Normal safety protocols are observed for experimental procedures. However, except for those normal procedures that the team can deal with on a daily basis, we will also use the ultraviolet light in our experiments so we decide to arrange different people to do this work to reduce the time contacting with it. As for the public and environment safety, we always keep the products in the cell cultures to avoid their spreading to the outside and check their conditions regularly. At the same time we will also dispose those products according to the protocols.<br><br>
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Fluorescence microscopy shows the preferential accumulation of VNP20009 pNirBeGFP in necrotic and hypoxic areas inB16F10 tumors. The nirB promoter could be induced specifically in this area but not in liver or spleen. (N, necrotic tumor area; V, vital tumorcells) This experiment was carried out in triplicate.
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We designed plasmid constructs expressinggenes of interest for Salmonella- mediated tumor-targeted therapy using the nirB promoter. Our fluorescence microscopy revealed that the nirB promoter could effectively drive theexpression of GFP in S. typhimuriumstrain VNP20009 carryingthe plasmid construct pNirBeGFP under hypoxia,suggesting that the nirB promoter was a potent promoter. <br/>
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S. typhimurium strain VNP20009 carryingpNirBeGFP or peGFP was grown for 24 h under anaerobic or aerobic conditions. Expression of GFP was examined by fluorescence microscope<br/><br/>
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Immunoblotting studies further showed that TRAIL was effectivelyexpressed in VNP20009 carrying the plasmid constructpTRAILgrowing under hypoxia.  <br/><br/>
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Bacteriallysates were prepared from VNP20009 carrying construct pTRAIL grown in anaerobic or aerobic jars and were subjected to immunoblottingassays using anti-TRAIL antibodies.
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These findings indicated thatgenes of interest for Salmonella- mediated tumor-targeted therapy could be effectively expressed in S. typhimurium strainVNP20009 carrying appropriate expression vectors driven bythe nirB promoter.
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Fluorescence intensity was determined. Bar represents the mean ± SD of five independent experiments (n =5;*P<0.001).
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Researches revealed that some auxotrophic strains can better colonize in tumors, indicating that special nutrient-rich environment can compensate for the nutrition the strains lack for. Therefore, we knocked out one gene in amino acid metabolism pathways of VNP.
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2.Do any of the new BioBrick parts (or devices) that you made this year raise safety issues?<br>
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  NO, none of the new BioBrick parts we made raises any significant safety issues since all the genes we use commonly exist in E.coli Top 10 and Salmonella typhimurium VNP. Although we give the transformed bacterias some advantages compared with the wild type, the advantages just exist in the tumor-targeting therefore it's still safe for us to use and manipulate. Given that we always keep the bacterias in the cell cultures, there is no chance for them to contact with the outside so it's harmless for the environment.
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3.Is there a local biosafety group, committee, or review board at your institution?<br>
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Yes, our team is supported by the Department of Life Science, Nanjing University. So we are under all the Department's safety regulations. Evaluating the experiments process in terms of safety, the department always reminds our team to put the safety to priority and arrange some experienced research assistants to help us.<br>
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4.Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?<br>
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  In terms of the safety issues, when the synthetic products have the chance to be commercial products, we should consider the harmony between synthetic and natural products and find an appropriate place for the synthetic products ,most importantly, establish some particular protocols for these products which are created by human.
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We found CFU (Colony Forming Units) of auxotrophic strains 5 and 6 could reach 109, while that of VNP can only reach 105 at most in normal tissues.  The ratio of number of bacteria in tumor tissues to that in liver on the 4th day shows that after knocking out related genes to the metabolism to synthesize certain amino acid, the ability of tumor targeting was highly enhanced, nearly 10 times compared to that of VNP.
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Latest revision as of 17:25, 26 September 2012

SafetyHome > results

To confirm the distribution of VNP20009 in mouse tissues and to verify the tumor-specific gene induction, we detected GFP expression in tumor, liver, and spleen by fluorescence microscopy. We found the VNPpLacZ-GFP (with constitutive promoter) could induce expression not only in necrotic tumor but also in spleen and liver tissue. However, VNPpNirBeGFP (with tumor specific promoter) could only be induced in hypoxic and necrotic tumor tissue.


Fluorescence microscopy shows the preferential accumulation of VNP20009 pNirBeGFP in necrotic and hypoxic areas inB16F10 tumors. The nirB promoter could be induced specifically in this area but not in liver or spleen. (N, necrotic tumor area; V, vital tumorcells) This experiment was carried out in triplicate.
We designed plasmid constructs expressinggenes of interest for Salmonella- mediated tumor-targeted therapy using the nirB promoter. Our fluorescence microscopy revealed that the nirB promoter could effectively drive theexpression of GFP in S. typhimuriumstrain VNP20009 carryingthe plasmid construct pNirBeGFP under hypoxia,suggesting that the nirB promoter was a potent promoter.


S. typhimurium strain VNP20009 carryingpNirBeGFP or peGFP was grown for 24 h under anaerobic or aerobic conditions. Expression of GFP was examined by fluorescence microscope



Immunoblotting studies further showed that TRAIL was effectivelyexpressed in VNP20009 carrying the plasmid constructpTRAILgrowing under hypoxia.



Bacteriallysates were prepared from VNP20009 carrying construct pTRAIL grown in anaerobic or aerobic jars and were subjected to immunoblottingassays using anti-TRAIL antibodies.
These findings indicated thatgenes of interest for Salmonella- mediated tumor-targeted therapy could be effectively expressed in S. typhimurium strainVNP20009 carrying appropriate expression vectors driven bythe nirB promoter.


Fluorescence intensity was determined. Bar represents the mean ± SD of five independent experiments (n =5;*P<0.001).
Researches revealed that some auxotrophic strains can better colonize in tumors, indicating that special nutrient-rich environment can compensate for the nutrition the strains lack for. Therefore, we knocked out one gene in amino acid metabolism pathways of VNP.


We found CFU (Colony Forming Units) of auxotrophic strains 5 and 6 could reach 109, while that of VNP can only reach 105 at most in normal tissues. The ratio of number of bacteria in tumor tissues to that in liver on the 4th day shows that after knocking out related genes to the metabolism to synthesize certain amino acid, the ability of tumor targeting was highly enhanced, nearly 10 times compared to that of VNP.