Team:UB Indonesia

From 2014.igem.org

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We try to make an idea to build a new prototype for early detection to cervical cancer, especially caused by HPV 18 and HPV 16. We construct a new specific TALEs that can bind to our target sequences and we called it TALE 1 and TALE 2. In order to make sure the presence of DNA  from HPV 16 and 18, we need reporter which connect to detector.  Fluorescent protein usually used as reporter gene to study about gene expression. It will be connected to detector using linker. When the HPV DNA is bind to detector, blue colour will appear in the second strip. The Intensity of colour will be detected in our software and we can determined the risk level of cervical cancer disease. Dark blue colour show that the amount of  DNA  from HPV 16 and 18 is high and indicate the patient get severe cervical cancer.<br><br>
We try to make an idea to build a new prototype for early detection to cervical cancer, especially caused by HPV 18 and HPV 16. We construct a new specific TALEs that can bind to our target sequences and we called it TALE 1 and TALE 2. In order to make sure the presence of DNA  from HPV 16 and 18, we need reporter which connect to detector.  Fluorescent protein usually used as reporter gene to study about gene expression. It will be connected to detector using linker. When the HPV DNA is bind to detector, blue colour will appear in the second strip. The Intensity of colour will be detected in our software and we can determined the risk level of cervical cancer disease. Dark blue colour show that the amount of  DNA  from HPV 16 and 18 is high and indicate the patient get severe cervical cancer.<br><br>
Beside that, we try to take the advantage of tea plant. Tea plant (Camelia sinensis) has catechin compounds that contains antioxidants.  Catechin family that most effective used as antioxidant is Epigallocathecin gallate (EGCG).  Based on research in vitro or in silico, EGCG is able to inhibit the proliferation of cancer cells because stop over-expression between L1 HPV 16- EGFR (Epithel Growth Factor Receptor) bond but there are few of EGCG content in tea plant. Based on these problems, we would to over- expression EGCG by knockdown non-compound EGCG gene, so hopefully the content of EGCG on tea are more prominent.<br><br>
Beside that, we try to take the advantage of tea plant. Tea plant (Camelia sinensis) has catechin compounds that contains antioxidants.  Catechin family that most effective used as antioxidant is Epigallocathecin gallate (EGCG).  Based on research in vitro or in silico, EGCG is able to inhibit the proliferation of cancer cells because stop over-expression between L1 HPV 16- EGFR (Epithel Growth Factor Receptor) bond but there are few of EGCG content in tea plant. Based on these problems, we would to over- expression EGCG by knockdown non-compound EGCG gene, so hopefully the content of EGCG on tea are more prominent.<br><br>
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CHS (Cancer Herbal Screening) is a kit which contains of hela cells as cervical cancer cells and it was designed to screening the plants that have compund potentially for cervical cancer therapy. Herbs that potentially as a therapy of cervical cancer in the kit are marked by the absence of green or red colors, while the negative results are marked by the emergence of green or red in the kit. The kit is expected to provide convenience to the researchers in the field of cervical cancer and cervical cancer effects can be mitigated with natural compound that not a lot of side effects. <br><br>
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CHS (Cancer Herbal Screening) is a kit which contains of hela cells as cervical cancer cells and it was designed to screening the plants that have compund potentially for cervical cancer therapy. Herbs that potentially as a therapy of cervical cancer in the kit are marked by the absence of green color, while the negative results are marked by the emergence of green in the kit. The kit is expected to provide convenience to the researchers in the field of cervical cancer and cervical cancer effects can be mitigated with natural compound that not a lot of side effects. <br><br>
From all of above, we bring C3P (Cervical Cancer Program) for iGEM 2014. We named our each project with preventing, screening and therapy. beside, we also do social action in other to socialize our project and be closer to society to inform them how to avoid cervical cancer. <br><br>
From all of above, we bring C3P (Cervical Cancer Program) for iGEM 2014. We named our each project with preventing, screening and therapy. beside, we also do social action in other to socialize our project and be closer to society to inform them how to avoid cervical cancer. <br><br>
The responsible of the scientist is not only the result in the lab, but also share “the right thing” for society. So, thanks to iGEM that gives us an opportunity for doing both, research and social action. <br><br>
The responsible of the scientist is not only the result in the lab, but also share “the right thing” for society. So, thanks to iGEM that gives us an opportunity for doing both, research and social action. <br><br>
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<b>Note:</b><br>
<b>Note:</b><br>
<table>
<table>
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<tr><td><img src="https://static.igem.org/mediawiki/2014/9/9a/UB-Check.PNG" width="52" height="43"></td><td>Done</td></tr>
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<tr><td><img src="https://static.igem.org/mediawiki/2014/9/9a/UB-Check.PNG" width="52" height="43"></td><td>=</td><td>Done</td></tr>
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<tr><td><img src="https://static.igem.org/mediawiki/2014/6/66/UB-Progress.PNG" width="52" height="43"></td><td>In Progress</td></tr>
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<tr><td><img src="https://static.igem.org/mediawiki/2014/6/66/UB-Progress.PNG" width="52" height="43"></td><td>=</td><td>In Progress</td></tr>
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<tr><td><img src="https://static.igem.org/mediawiki/2014/f/f3/UB-Silang.PNG" width="52" height="43"></td><td>Not yet</td></tr>
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<tr><td><img src="https://static.igem.org/mediawiki/2014/f/f3/UB-Silang.PNG" width="52" height="43"></td><td>=</td><td>Not yet</td></tr>
</table>                                         
</table>                                         
                                         </div>
                                         </div>
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<p align="justify">
<p align="justify">
<b>Overview Therapy</b><br>
<b>Overview Therapy</b><br>
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CHS (Cancer Herbal Screening) part is design plasmid which contain  promoter P97 (HPV 16) or P105 (HPV 18). This part has ability in screening chemical compounds of plant for cervical cancer therapy. The herbs have to inhibit transcription factor of P97 and P10. Promoter was combained with fluorescence reporter GFP obtained from biobrick Bba_E0240 (Plate 4, well 11N) to detect the successfull of the herbal compounds.  P97 (HPV 16) and P105(HPV 18) were digested with EcoR1-Xbal, GFP digested with Xbal-Spel, and plasmid backbone PSB1C3 digested with EcoR1-Spel. Then, the HPV promoter and GFP was ligated into palsmid backbone PSB1C3. Plasmid design was transfected into HeLa cells containing some transcripton factor to induced promoter. This part was purposed to help researcher work easier to explore herbal plants for cervical cancer therapy.<br>
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Cervical cancer is the one of most killing cause of woman. Approximately, 300.000 people deaths by cervical cancer in every year (Frazer, Cox et al., 2006; Lowy and Schiller, 2006).Cervical cancer caused by Human papillomaviruses (HPV) (Martinez et al., 2008).More than 100 types of HPVs have been identified to date (Dell et al., 2001), but the two most common types in cervical cancer are HPV 16 and 18. HPV type 16 and 18 are high risk type that causecervical cancer (Das, 2003). Both of those type responsible for approximately 70 % of all cases (Smith et al., 2007).<br><br>
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<center><img src="https://static.igem.org/mediawiki/2014/5/52/UB-construct-hps.PNG" width="500" height="300"></center>   
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HPV are small double-stranded DNA viruses that contain of two oncogenes E6 and E7 that are involved in cellular transformation. Two viral genes, E6 and E7 are commonly found expressed in these cancer cells (Song, 2000). Transcription of the human papillomavirus type 16 (HPV-16) genome is controlled by several promoters and the P97 promoter is considered to be the main one (Braunstein et al., 1999). P97 is the HPV-16 early promoter involved in the transcription of E6, E7 and other viral genes (Dell and Gaston, 2011; Flores and Lambert, 1997; Hebner and Laimins, 2006. Based on Laura et al (2005), P105 is located to the next E6 start codon and is the main early promoter of HPV-18. AP-1, Sp-1, NF-1, Oct-1, TEF-1, YY-1, KRF-1, Skn-1a, and TFIID known as transcription factors that could stimulate P105 promoter activity. HPV-18 P105 promoter and HPV-16 P97 promoter has equivalent function. E2 repression of P105 promoter activity has been demonstrated in HeLa cells (Thierry and Yaniv, 1987) and in primary human keratinocytes (Bernard et al., 1989). Romanczuk et al(1990), suggested that P97 and P105 promoters both could be regulated by similar mechanisms. A comparison of the sequences upstream of the HPV-16 P97 promoter and the HPV-18 P105 promoter revealed a similar spatial arrangement of the four E2-binding sites, the TATAAAA boxes, and the start sites for transcription in the respective viral LCRs. (Romanczuk et al, 1990).  Based on Chong et al(1991), trancript factor such as NF1, AP-1, and oct-1 were present in higher concentration in HeLa cells. Therefor, HeLa cells can be used as transfection for promoter P97 and promoter P105 so it could be activated.<br><br>
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Indonesia has many potential herb that potentially used as medicine for cervical cancer.  The major goal ofthis research is to create a kit that can screen herb potentially used as medicine for cervical cancer. It can give early information for researcher, doctor and other practical that can be used as reference to continue the research as well as to give scientific. CHS (Cancer Herbal Screening) part is design plasmid which contain  promoter P97 (HPV 16) or P105 (HPV 18). This part has ability in screening chemical compounds of plant for cervical cancer therapy. The herbs have to inhibit transcription factor of P97 and P10. <br><br>
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 +
Promoter was combained with fluorescence reporter GFP obtained from biobrick Bba_E0240 (Plate 4, well 11N) to detect the successfull of the herbal compounds.  P97 (HPV 16) and P105 (HPV 18) were digested with EcoR1-Xbal, GFP digested with Xbal-Pstl, and plasmid backbone pSB1C3 digested with EcoR1-Pstl. Then, the HPV promoter and GFP was ligated into palsmid backbone pSB1C3. Plasmid design was transfected into HeLa cells containing some transcripton factor to induced promoter. Based on Chong et al (1991), trancript factor such as NF1, AP-1, and oct-1 were present in higher concentration in HeLa cells. Therefor, HeLa cells can be used as transfection for promoter P97 and promoter P105 so it could be activated. This part was purposed to help researcher work easier to explore herbal plants for cervical cancer therapy.</p> <br><br>
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<center><img src="https://static.igem.org/mediawiki/2014/e/ef/UB-overview_therapy.PNG" width="500" height="300"><br>
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Figure 1. Construction of CHS (Cancer Herbal Screening) in pSB1C3
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</center>   
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<p align="justify">
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<b>References :</b><br>
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<ul>
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<li>Bernard, B. A., C. Bailly, M.-C. Lenoir, M. Darmon, F. Thierry, and M. Yaniv. 1989. The human papillomavirus type 18 (HPV18) E2 gene product is a repressor of the HPV18 regulatory region in human keratinocytes. J. Virol  63:4317-4324.</li>
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<li>Braunstein TH, BS Madsen, B Gavnholt, MW Rosenstierne, C Koefoed Johnsen and B norrild. 1999. Identification of a new promoter in the early region of the  human papillomaviruses type 16 genome. Journal of General Virology 80: 3241-3250</li>
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<li>Chong, T., D. Apt, B. Gloss, M. Isa, and H. U. Bernard. 1991. The enhancer of human papillomavirus type 16: binding sites for the ubiquitous transcription factors Oct-1, NFA, TEF-2, NF1, and AP1 participate in epithelial cell-specific transcription. J. Virol. 65:5933–5943. </li>
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<li>Das, B.C. 2003. Cellular control of human papillomavirus gene activity in cervical cancer. Proc.Indian natn Sci Acan. B69 No.1 pp23-24. </li>
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<li>Dell, G., and K. Gaston. 2001. Contributions in the domain of cancer research:review of human papillomaviruses and their role in cervical cancer.Cell. Mol. Life Sci.58:1923–1942. </li>
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<li>Flores, E. R., and P. F. Lambert. 1997. Evidence for a switch in the mode of human papillomavirus type 16 DNA replication during the viral life cycle. J. Virol. 71:7167–7179. </li>
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<li>Frazer, I. H., J. T. Cox, et al. 2006. Advances in prevention of cervical cancer and other human papillomavirus-related diseases.Pediatr Infect Dis J 25(2 Suppl): S65-81, quiz S82. </li>
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<li>Hebner, C. M., and L. A. Laimins. 2006. Human papillomaviruses: basic mechanisms of pathogenesis and oncogenicity. Rev. Med. Virol16:83–97. </li>
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<li>Laura Sichero, Eduardo Luis Franco, and Luisa Lina Villa. 2005. Different P105 Promoter Activities among Natural Variants of Human Papillomavirus Type 18. The Journal of Infectious Diseases191:739–42. </li>
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<li>Lowy, D. R. and J. T. Schiller. 2006. Prophylactic human papillomavirus vaccines. J Clin Invest 116(5): 1167-73. </li>
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<li>Martinez., AS Gardiner, KF Board, FA Monzon, RP Edwards and SA Khan. 2008. Human papillomavirus type 16 reduces the expression of microRNA-218 in cervical carcinoma cells. Oncogene 27:2575-2582</li>
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<li>Romanczuk H., F Thierry and P M Howley. 1990.Mutational analysis of cis elements involved in E2 modulation of human papillomavirus type 16 P97 and type 18 P105 promoters. J. Virol64(6):2849. </li>
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<li>Smith JS, Lindsay L, Hoots B, Keys J, Franceschi S, Winer R, et al. 2007. Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. International journal of cancer121:621-32. </li>
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<li>Song, Shiyu., Amy Liem, James A., Miller and Paul F Lambert. 2000. Human Papillomavirus Types 16 E6 and E7 Contribute Differently to Carcinogenesis. Virology 267:141-150. </li>
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<li>Thierry, F., and M. Yaniv. 1987. The BPV1-E2 trans-acting protein can be either an activator or a repressor of the HPV 18 regulatory region. EMBO J6:3391-3397. </li>
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<li>Thierry, F., J. M. Heard, K. Dartmann, and M. Yaniv. 1987. Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens. J. Virol61:134-142. </li>
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</ul>
</p>
</p>
                                         </div>
                                         </div>
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<ul>
<ul>
<li>Week 1</li>
<li>Week 1</li>
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Splitting HeLa cells. The confluence HeLa cells (80 – 90%) splitting into another flask then incubate in 37ᵒC.
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Splitting HeLa cells. The confluence HeLa cells (80 – 90%) splitting into another flask then incubate in 37ᵒC.<br>
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<center>https://static.igem.org/mediawiki/2014/0/0f/UB-HELA.jpg</center>
<li>Week 2</li>
<li>Week 2</li>
Cell observation. The splitting HeLa cells observed to control the growth.
Cell observation. The splitting HeLa cells observed to control the growth.
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<table width="100%">
<table width="100%">
<tr><td>Discussion about the material and tool which complete the task of the devices. We  looked for some well known, user friendly, easy handle, effective, and efficient. We also get some material recommendation and suggestion for our device. Then, by some review and consideration, we decided to use a test pack for the tool (hardware) and colour intensity as the diagnostic result.</td>
<tr><td>Discussion about the material and tool which complete the task of the devices. We  looked for some well known, user friendly, easy handle, effective, and efficient. We also get some material recommendation and suggestion for our device. Then, by some review and consideration, we decided to use a test pack for the tool (hardware) and colour intensity as the diagnostic result.</td>
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<td><img src="" width="50%" height="100"></td></tr>
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<td><img src="https://static.igem.org/mediawiki/2014/4/49/Ub-Test_pack_1.jpg" width="400" height="200"></td></tr>
</table><br>
</table><br>
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<b>August 4th – August 8th (Week 2)</b><br>
<b>August 4th – August 8th (Week 2)</b><br>
<table width="100%">
<table width="100%">
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<tr><td><img src="" width="50%" height="100"></td>
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<tr><td><img src="https://static.igem.org/mediawiki/2014/5/56/UB-Test_pack_2.jpg" width="400" height="150"></td>
<td>Chose and bought some test pack to be learned. Here, we got some model of test pack to study and determine the model from one of common trademark as the basic model of ours.</td></tr>
<td>Chose and bought some test pack to be learned. Here, we got some model of test pack to study and determine the model from one of common trademark as the basic model of ours.</td></tr>
</table><br>
</table><br>
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We try to put the enzyme into model and observed the result. We performed some test in different variable to determine its reliability.<br>
We try to put the enzyme into model and observed the result. We performed some test in different variable to determine its reliability.<br>
<table width="100%">
<table width="100%">
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<tr><td><img src="" width="50%" height="100"></td>
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<tr><td align="right"><img src="https://static.igem.org/mediawiki/2014/d/db/UB-TP_1.jpg" width="300" height="100"></td>
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<td><img src="" width="50%" height="100"></td></tr>
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<td><img src="https://static.igem.org/mediawiki/2014/8/8c/UB-TP_2.jpg" width="300" height="100"></td></tr>
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<tr><td><img src="" width="50%" height="100"></td>
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<tr><td align="right"><img src="https://static.igem.org/mediawiki/2014/0/02/UB-TP_3.jpg" width="300" height="100"></td>
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<td><img src="" width="50%" height="100"></td></tr>
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<td><img src="https://static.igem.org/mediawiki/2014/7/77/UB-TP_4.jpg" width="300" height="100"></td></tr>
</table><br>
</table><br>
<b>September 15th – September 19th (Week 3)</b><br>
<b>September 15th – September 19th (Week 3)</b><br>
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Still continue to test the device and get more result.<br>
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Still continue to test the device and get more result.<br><br>
<b>September 22nd – September 26th (Week 4)</b><br>
<b>September 22nd – September 26th (Week 4)</b><br>
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Get some suggestion and more explanation from our lecturer in Biology about the result <br>
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Get some suggestion and more explanation from our lecturer in Biology about the result <br><br>
<b>September 29th – October 3rd </b><br>
<b>September 29th – October 3rd </b><br>
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Performed stability test to know more about the device. We even did it with a little various differentiation.<br>
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Performed stability test to know more about the device. We even did it with a little various differentiation.<br><br>
<b>October 2th – October 5th  (Week 1)</b><br>
<b>October 2th – October 5th  (Week 1)</b><br>
We designed the software content and review it from some journals and articles.<br>
We designed the software content and review it from some journals and articles.<br>
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<center>
 +
<img src="https://static.igem.org/mediawiki/2014/c/cf/Ub-software1.jpg" width="300" height="500"><br><br>
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<img src="https://static.igem.org/mediawiki/2014/1/1e/Ub-software2.jpg" width="300" height="500"><br><br>
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<img src="https://static.igem.org/mediawiki/2014/d/da/Ub-software3.jpg" width="300" height="500"><br><br>
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<img src="https://static.igem.org/mediawiki/2014/4/41/Ub-software4.jpg" width="300" height="500"><br><br>
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<img src="https://static.igem.org/mediawiki/2014/c/c9/Ub-software5.jpg" width="300" height="500"><br><br>
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</center>
<b>October 6th – October 10th  (Week 2)</b><br>
<b>October 6th – October 10th  (Week 2)</b><br>
Collected the results and put it into results table to be presented.<br>
Collected the results and put it into results table to be presented.<br>
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We try to develop the software with ECLIPSE program .<br>
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We try to develop the software with ECLIPSE program .<br><br>
</p>
</p>
</ul>
</ul>
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<li> Promoter CMV in plate 1 well 21P inserted into PSB1C3 with chloramphenicol resistance.</li>
<li> Promoter CMV in plate 1 well 21P inserted into PSB1C3 with chloramphenicol resistance.</li>
</ul>
</ul>
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"We acknowledge to all members of IGEM ITB team for helping us doing of parts project."
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"We acknowledge to all members of IGEM ITB team for helping us doing of parts project."<br><br>
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For developing TALE 1 and TALE 2, we've got the material- GATE Assembly Kit, pTALEN and pTAL-TF -from BIOSS at University of Freiburg by Nicole Gensch. <br>
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<img src="https://static.igem.org/mediawiki/2014/d/dd/UB-Bioss.jpg" height="200" width="400">
                                         </div>
                                         </div>
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<a href="#fav-parts" class="fancybox"><p><i class="icon icon-group"></i></p></a>
<a href="#fav-parts" class="fancybox"><p><i class="icon icon-group"></i></p></a>
                                         <div id="fav-parts" style="display:none;width:700px;">
                                         <div id="fav-parts" style="display:none;width:700px;">
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<h2><center>“”</center></h2>
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<h2><center>Favorite Parts</center></h2><br>
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<p align="justify"></p>
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<a href="http://parts.igem.org/Part:BBa_E0240:Design"><b>Bba_E0240 GFP_Generator </b></a><br><br>
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<center>
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<img src="https://static.igem.org/mediawiki/2014/0/0b/UB-GFP_1.PNG" width="200" height="100"><br>
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<img src="https://static.igem.org/mediawiki/2014/d/d9/UB-GFP-PLASMID_2.PNG" width="500" height="500"></center><br><br>
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<a href="http://parts.igem.org/Part:BBa_K747012"><b>Bba_K747012 TALE_Protein_TA1_Direpeat </b></a><br><br>
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<center><img src="https://static.igem.org/mediawiki/2014/9/9b/UB-TALE-PLASMID_2.PNG" width="500" height="500"></center>
                                         </div>
                                         </div>
<h2 class="font-thin"><font color="#fff">Favorite Parts</font></h2>
<h2 class="font-thin"><font color="#fff">Favorite Parts</font></h2>

Latest revision as of 01:15, 18 October 2014

iGEM2014 | UB INDONESIA

BRAWIJAYA UNIVERSITY , INDONESIA

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