Difference between revisions of "Team:NCTU Formosa/Safety"

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  <div class="project">
 
<div class="Background"><a class="link link--kukuri" href="#" data-letters="Safety"> Safety</a></div>
 
 
 
 
<div class="project_text" style="text-align:justify;">
 
    <p>This year Apollo designed a product mainly to detect antigens. Our users include scientific researchers, clinical scientist and so on. We believe these people all have the expertise, but we would like to once again remind everyone that our product contains <I>E. coli</I>. Therefore when applying our product, basic protective gear (lab coat, gloves and so on) are needed. Objects that come into contact with our product (for example: tips) need to be sterilized and thrown away, in case of non-natural gene outflow. We’ll hand our product to others after our safety procedure. Hence lowering the possibility of bio-contamination. </P>
 
<p>We designed a safety mechanism for our E.Cotector allowing it to be friendlier to users and environment. Our main objective is to maintain the fluorescence but kill the bacteria, which means that treated <I>E. coli</I> cannot grow in nutritious conditions such as LB.</p></div>
 
<div class="project_title">
 
<strong>Choice</strong></div>
 
<div class="project_text"style="text-align:justify;">
 
<p><B>Paraformaldehyde</B></p>
 
<p>At first, we treated our E.Cotector, displayed scFv(anti-EGFR) and green fluorescence protein(GFP), by paraformaldehyde. Paraformaldehyde is a chemical substance that is toxic for organism. It can fix the cell structure and all of the proteins.</p>
 
  
<p><B>Antibiotic</B></p>
 
<p>We tried antibiotics to achieve our goal. We used three kinds of antibiotics. The first one is tetracycline. It can bind to 30S subunit of ribosomes and then inhibit the synthesis of proteins. The second one is ampicillin. It can inhibit the formation of the cell wall. The third one is sulfonamide (p-Aminobenzenesulfonamide). It competitively inhibits the synthesis of folate, which connects to the purine synthesis and the DNA synthesis <SUP>[3] [4]</SUP>.</p>
 
<p><B>Result</B></p>
 
<p>We mixed the antibiotic and bacteria together. Then we observed the growth and the fluorescence of bacteria. There will be more details in the protocol.
 
The category and testing concentration of antibiotic are bellowed.</p>
 
<p>In the experiment where we add Tetracycline and Ampicillin, we discovered the more antibiotics we added the less the fluorescence(Figure 1). Tetracycline reduces the fluorescence at a higher rate than Ampicillin. But when we cultivate the bacteria on LB plate, we noticed that the whether we add Ampicillin or not, the bacteria still grows on the plate. On the other hand, bacteria added with Tetracycline won’t grow on the LB plate(Figure 2). </p>
 
<div style="text-align:center;font-size:12pt;"><img style="margin:0 auto;" src="https://static.igem.org/mediawiki/2015/1/1c/NCTU_Formosa_Safety1.png" width="760px" height="529.5px">
 
<p>Figure 1. Impacted by ampicillin and tetracycline,there were the expression of fluorescence during 12 hours.</p></div>
 
<div style="text-align:center;font-size:12pt;"><img style="margin:0 auto;" src="https://static.igem.org/mediawiki/2015/2/26/NCTU_Formosa_Safety3.png" width="618.7px" height="360px">
 
<p>Figure 2. The result of incubated the E.Cotector on the LB plate treated by ampicillin or tetracycline.<p></div>
 
<p>
 
In the next experiment where we add sulfonamide, we observed that the fluorescence is more or less the same amount as the original bacteria(Figure 3). Correspondingly, we also cultivated the cell on LB plate. We discovered the inhibition of growth doesn’t steadily change with the concentration of Sulfonamide. Some bacteria still grow and some don’t(Figure 4). </p>
 
<div style="text-align:center;font-size:12pt;"><img style="margin:0 auto;" src="https://static.igem.org/mediawiki/2015/3/3c/NCTU_Formosa_Safety2.png" width="760px" height="529.5px">
 
<p>Figure 3. Impacted by sulfulnamide,there were the expression of fluorescence during 12 hours.</p></div>
 
<div style="text-align:center;font-size:12pt;"><img style="margin:0 auto;" src="https://static.igem.org/mediawiki/2015/6/6c/NCTU_Formosa_Safety4.png" width="618.7px" height="352px">
 
<p>Figure 4. The result of incubated the E.Cotector on the LB plate treated by sulfonamide.<p></div>
 
<p><B>Conclusion</B></p>
 
<p>From the experiment, we learned that different antibiotics have different amounts of fluorescence conservation. The conservation effect: Ampicillin>Sulfonamide>Tetracycline. The conservation amounts are all sufficient for detection. Furthermore, the growth condition of our bacteria treated by different antibiotics also varies. The inhibition effect: Tetracycline>Sulfonamide>Ampicillin. Among the three, Sulfonamide’s effect is unstable. Might be because of Sulfonamide’s solubility is highly related to the solution’s pH value which causes the solubility to decrease causing the concentration of Sulfonamide to distribute unequally. We put the result of adding different antibiotics in Table 1. Lastly, since collectively Tetracycline performed best at killing the bacteria and conserving the fluorescence, we chose tetracycline to continue our safety.  </p>
 
<div style="text-align:center;font-size:12pt;"><img style="margin:0 auto;" src="https://static.igem.org/mediawiki/2015/1/1f/NCTU_Formosa_Safety5.png" width="1044px" height="509.25px">
 
<p>Table 1. Comparison of different antibiotics effected on the E.Cotector.</p></div>
 
</div>
 
<div class="project_title">
 
<strong>Best concentration and reaction time</strong></div>
 
<div class="project_text"style="text-align:justify;">
 
<p>Since we chose tetracycline as our main focus, we need to test how much concentration of tetracycline can achieve the best sterilization effect. Furthermore, we want to learn how long does it take to let the antibiotic work efficiently so that it cannot grow on the LB plate. We add different concentration of tetracycline to our E. cotector and compare it with E. cotector without tetracycline. After adding the antibiotic, we extracted appropriate amount of bacterial liquid from each sample and added to 96 well to detect the fluorescence. In the mean time, we also extracted 1 uL of bacterial liquid from each sample and added on to LB plate. We repeat this step every hour and continue for 7 hours. Moreover, we cultivate the LB plate long enough to make sure all the living bacteria will grow. We observe the best concentration and best reaction time for tetracycline to effectively kill the bacteria.</p>
 
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        <div style=float:left;> <a href="https://2015.igem.org/Team:NCTU_Formosa/Design" class="btn btn-sm animated-button thar-three">Go to Design</a> </div>
 
<div style=float:left;> <a href="https://2015.igem.org/Team:NCTU_Formosa/Project" class="btn btn-sm animated-button thar-three-two">Back to project</a> </div>
 
           
 
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    <div style="width:30vw;height:5vh;position:absolute;left:2vw;float:left;background-color:#00171F;color:#fff;font-family:Myriad Pro;font-size:2em;padding:7vh 0vw 7vh 5vw;">contact us<BR> NCTU_Formosa APOllO &nbsp;&nbsp;<a href="https://www.facebook.com/pages/NCTU_Formosa-IGEM-team/267841893250331?fref=ts" target="_blank"><img src=https://static.igem.org/mediawiki/2015/a/ab/NCTU_FORMOSA_Facebook_LINK.png width=40vw></a> </div>
 
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            &nbsp;&nbsp;Engineering Building 6 EF455, 1001 University Road, Hsinchu 300, Taiwan, ROC. </div>
 
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<div class="title">Safety</div>
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<div class="contentitle">
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Single chain variable fragment as probe
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    Single chain variable fragment (scFv) Abs are one of the <font color=#b51c48> recombinant antibody(rAb)</font> fragments, which are popular therapeutic alternatives to full length of monoclonal Abs. Compared to generating whole Abs from animal cell culture, scFv are smaller and can be expressed rapidly, economically and in large quantities in a bacterial host, such as<font color=#b51c48> E. coli</font>. A scFv <font color=#b51c48>possesses the complete antigen binding site</font>, which contains the variable heavy (VH) and variable light domain of an antibody. The VH domain is linked to a VL domain by an introduced flexible polypeptide linker. A scFv is capable of binding its target antigens with an affinity similar to that of the parent mAb. Due to containing the specific antigen binding unit, scFv fragments show tremendous versatility and importance in<font color=#b51c48> human therapeutics and diagnostics</font>. [1] In addition, scFv fragments can be envisaged to be applied in the non-pharmaceutical sector, such as in the food, cosmetic or environmental industries. The unique and highly specific antigen-binding ability might, for example, be exploited to block specific enzymes (e.g. enzymes that cause food spoilage), bacteria (e.g. in toothpaste or mouthwashes) or to detect environmental factors present in very low concentrations (as biosensors).[2]
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<div class="contentitle">Properties and development of targeted drugs</div>
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<div class="content">
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This year, we decided to utilize the scFv as probes to detect cancer markers and aid in the prescription of targeted drugs in cancer treatments.
 +
Targeted drugs therapy utilize compounds that are capable of inhibiting target molecules, the cancer markers which send messages along signaling pathways in cell growth, cell division or cell death. Via specific binding to target molecules, targeted drugs show more accurate attack to cancer cells and less harmful damage to normal tissues. [1] The precision of targeting the cancer cells has enhanced the efficiency of treatment by a large margin. The targeted therapy is a major step forward for many cancers, especially advanced cancers, and physicians and researchers are now focusing on the development of targeted drugs, creating a new era of personalized cancer treatment.[3]Targeted therapy are so-called "personalized medicine" because health care professionals can use clinical test results from a patient to select a specific drug that has a higher likelihood of being effective for that particular person.<br><br>
 +
According to the statistics, the usage rate of targeted drug therapy has increased within ten years. In Figure 1, in 2003, targeted drug therapy is not commonly used compared with other therapies, accounting for only 11% usage. Over one decade, it is estimated that the usage of targeted drug therapy dramatically increases to<font color=#b51c48> 46%</font>. It indicates targeted drugs therapy is a potential growing field and will become the commonly used therapy in cancer treatments in the near future.
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<br><br>
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<div class="contentitle">Pre-diagnosis of targeted drugs treatment</div>
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<div class="content">
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To create the new era of tailored targeted drugs, doctors must aim at<font color=#b51c48> appropriate target molecules </font>for patients with particular diseases. In 2014,<font color=#b51c48> the U.S. Food and Drug Administration (FDA) </font>issued a guidance to facilitate the development and review of <font color=#b51c48>diagnostics tests</font>. The diagnostics tests are the steps to identify the abnormal cancer biomarkers. Moreover, the purpose of diagnostics tests are to help medical practitioners <font color=#b51c48>determine which patients could benefit from the certain drugs</font>, conversely, those who should not receive the medication. If the treatment decisions is not optimal, it would not only cause the fatal body damage, but also lead to the waste of time, money and medical resources. FDA encourages the joint of targeted drugs therapies and precise diagnostics tests which are essential for the safe and effective use of targeted drugs.[4]
 +
</div>
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<div class="contentitle">The concept of combination therapy</div>
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<div class="content">
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Although targeted drugs treatments can lead to the dramatic regressions of solid tumors, the responses are often short-lived because resistant cancer cells arise after a period of treatment. The major strategy proposed for overcoming the resistance is <font color=#b51c48>combination therapy</font>. The clinical and preclinical researches further indicated that targeted drug therapy combined with another targeted drug therapy or other types of therapies to treat cancers simultaneously may attain greater effects than using only one therapy. With the concept of combination therapy, we can not only improve the treating effect but also reduce the occurrence of cancer cells resistance toward the targeted drugs as there are less probability that a single mutation will cause cross-resistance to both drugs.[2] </div>
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<div class="contentitle">APPOllO E.Cotector</div>
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<div class="content">To enhance the <font color=#b51c48>efficiency of diagnosis </font>and provide reference for<font color=#b51c48> proper usage of targeted drugs</font> and <font color=#b51c48>combination therapy</font>, we come up with the idea of detecting multimarker at the same time and this was how our marvelous E.Cotector is borned. This year, NCTU_Formosa commits to creating a multimarker diagnosis platform via scFv as probes for helping physicians to determine and prescribe the usage of targeted drugs in cancer patients, especially the monoclonal-antibody-targeted drugs.</div>
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Revision as of 17:30, 8 September 2015

Safety
Single chain variable fragment as probe
Single chain variable fragment (scFv) Abs are one of the recombinant antibody(rAb) fragments, which are popular therapeutic alternatives to full length of monoclonal Abs. Compared to generating whole Abs from animal cell culture, scFv are smaller and can be expressed rapidly, economically and in large quantities in a bacterial host, such as E. coli. A scFv possesses the complete antigen binding site, which contains the variable heavy (VH) and variable light domain of an antibody. The VH domain is linked to a VL domain by an introduced flexible polypeptide linker. A scFv is capable of binding its target antigens with an affinity similar to that of the parent mAb. Due to containing the specific antigen binding unit, scFv fragments show tremendous versatility and importance in human therapeutics and diagnostics. [1] In addition, scFv fragments can be envisaged to be applied in the non-pharmaceutical sector, such as in the food, cosmetic or environmental industries. The unique and highly specific antigen-binding ability might, for example, be exploited to block specific enzymes (e.g. enzymes that cause food spoilage), bacteria (e.g. in toothpaste or mouthwashes) or to detect environmental factors present in very low concentrations (as biosensors).[2]
Properties and development of targeted drugs
This year, we decided to utilize the scFv as probes to detect cancer markers and aid in the prescription of targeted drugs in cancer treatments. Targeted drugs therapy utilize compounds that are capable of inhibiting target molecules, the cancer markers which send messages along signaling pathways in cell growth, cell division or cell death. Via specific binding to target molecules, targeted drugs show more accurate attack to cancer cells and less harmful damage to normal tissues. [1] The precision of targeting the cancer cells has enhanced the efficiency of treatment by a large margin. The targeted therapy is a major step forward for many cancers, especially advanced cancers, and physicians and researchers are now focusing on the development of targeted drugs, creating a new era of personalized cancer treatment.[3]Targeted therapy are so-called "personalized medicine" because health care professionals can use clinical test results from a patient to select a specific drug that has a higher likelihood of being effective for that particular person.

According to the statistics, the usage rate of targeted drug therapy has increased within ten years. In Figure 1, in 2003, targeted drug therapy is not commonly used compared with other therapies, accounting for only 11% usage. Over one decade, it is estimated that the usage of targeted drug therapy dramatically increases to 46%. It indicates targeted drugs therapy is a potential growing field and will become the commonly used therapy in cancer treatments in the near future.


Pre-diagnosis of targeted drugs treatment
To create the new era of tailored targeted drugs, doctors must aim at appropriate target molecules for patients with particular diseases. In 2014, the U.S. Food and Drug Administration (FDA) issued a guidance to facilitate the development and review of diagnostics tests. The diagnostics tests are the steps to identify the abnormal cancer biomarkers. Moreover, the purpose of diagnostics tests are to help medical practitioners determine which patients could benefit from the certain drugs, conversely, those who should not receive the medication. If the treatment decisions is not optimal, it would not only cause the fatal body damage, but also lead to the waste of time, money and medical resources. FDA encourages the joint of targeted drugs therapies and precise diagnostics tests which are essential for the safe and effective use of targeted drugs.[4]
The concept of combination therapy
Although targeted drugs treatments can lead to the dramatic regressions of solid tumors, the responses are often short-lived because resistant cancer cells arise after a period of treatment. The major strategy proposed for overcoming the resistance is combination therapy. The clinical and preclinical researches further indicated that targeted drug therapy combined with another targeted drug therapy or other types of therapies to treat cancers simultaneously may attain greater effects than using only one therapy. With the concept of combination therapy, we can not only improve the treating effect but also reduce the occurrence of cancer cells resistance toward the targeted drugs as there are less probability that a single mutation will cause cross-resistance to both drugs.[2]
APPOllO E.Cotector
To enhance the efficiency of diagnosis and provide reference for proper usage of targeted drugs and combination therapy, we come up with the idea of detecting multimarker at the same time and this was how our marvelous E.Cotector is borned. This year, NCTU_Formosa commits to creating a multimarker diagnosis platform via scFv as probes for helping physicians to determine and prescribe the usage of targeted drugs in cancer patients, especially the monoclonal-antibody-targeted drugs.