document.getElementById("isi").innerHTML = "<img src='https://static.igem.org/mediawiki/2015/2/24/UI_INDONESIA_GRANDDESIGN.png' width='500px'><br>We design a device for gram positive bacteria. After discussion, we chose to use B. subtilis 168and E. coli BL21as our host. B. subtilis 168 is a model for gram positive toggle switch. E. coli BL21 and B. subtilis 168 are chosen for chassis for expression of SboA.<br>For grand design we designeda toggle switch with the spermicidal protein; Subtilosin A (SboA) in the system. In the other side of the switch, we designed a suicide protein: NdoA.<br>The promoter in the system are Phyperspank(Phs) and PxylA. Phyperspank is a constitutive promoter  for gram positive when the repressor protein, LacI is not present. When present, LacI bound to Phs, inhibiting the expression of the promoter. However, with addition of lactose or IPTG, LacI would bind to IPTG and detach from the promoter, causing the promoter to express the protein downstream.<br>PxylA is another constitutive promoter for gram positive. PxylA is repressed by xylR. Xylose would bind to xylR and cause PxylA to express the protein downstream.<br>We express xylR under the effect of Phs and express LacI under the effect of PxylA. Thus, when one promoter is active, it repress the other promoter. When PxylA is active, it express SboA, the spermicidal protein. It also express LacI which inhibit the other promoter, Phs. When induced with IPTG, LacI binds to IPTG and inhibition on Phs decrease. Thus protein downstream of Phs active, which is XylR. XylR repress PxylA, thus the expression of SboA stop, and the production of NdoA started.<br>This Grand Design was the ideal design. However, we are not testing this device as a whole due to safety reason. The safety reason issue is the pathogenicity of the bacteria if we test this device as a whole.<br><br><b>SboA Expression</b><br><img src='https://static.igem.org/mediawiki/2015/7/74/UI_Indonesia_Granddesign2.png' width='200px'><br>For characterization of SboA in E. coli, we use pET9a with T7 promoter. The plasmid was transformed to E. coli BL21 CP. Culture was grown overnight. 1/10 of the culture was transferred to TB medium of 5 mL. It was shaken for 2 hours. 500μL of the culture was taken as a control before induction, the rest was induced with final concentration of 1mM IPTG. Culture of 2 hours and 4 hours after induction was taken and centrifuged. The pellet and the supernatant was taken.<br><br><b>Toggle Switch</b><br><br><img src='https://static.igem.org/mediawiki/2015/b/b6/UI_Indonesia_ToggleSwitvch.png' width='500px'><br>To characterize the toggle switch, we switch the SboA with GFP and NdoA with RFP. Single colony was streaked over LB agar with antibiotic. After 18 hours, the culture was induced with 1M IPTG and observed under UV.<br>Other characterization method involve culture of overnight colony to TB broth. After 2 hours shaking, the culture was induced with 1mM final concentration of IPTG. Another culture was induced with 1% xylose. The result was observed under UV.<br><br><b>Method</b><br>a. Sperm Motility Assay<br>Sperm Motility Assay was performed to see the engineered subtilosin A effect on human sperm<br>For motility assay, we use human spermatozoa. Ethical clearance was issued. Subject was informed and consented.<br>Sample was collected by self masturbation. It was immediately (within 1 hour) observed under light microscope. On counting sperm motility, we ask professional help from Departement of Biology Universitas Indonesia. The method was random, blind study.<br>Modified Sutyak KE, et al was used to determine the sperm motility assay. 200μL of culture was mixed with 40μL of whole semen.  The percentage of forward moving spermatozoa was documented.<br><br>b. Well Diffusion Assay<br>Well diffusion assay was performed to see Subtilosin A effectivity as bacteriocin.A 3 hour culture of M. luteus in Nutrient broth was spread in blood agar and Mueller Hinton Aga.<br><br><b>Result</b><br>a. Sperm Motility Assay<br>Decrease in sperm motility was observed after intervention. We add Media+antibiotic as a control for observing sperm death due to addition of media +antibiotic (TB+tetracycline).C1, C2, and C3 was culture 1,2, and 3 respectively. WT represent wild type BL21 CP without plasmid.<br><img src='https://static.igem.org/mediawiki/2015/f/f6/Ui_indonesia_tabel1oi.png' width='400px'><br>Sperm Motility Assay after Intervention<br><img src='https://2015.igem.org/File:Ui_indonesia_tabel2oi.png' width='400px'><br>Sperm motility compared to wild type<br><img src='https://static.igem.org/mediawiki/2015/7/7c/Ui_indonesia_tabel3oi.png' width='400px'><br><br>Graph explanation<br>Y bar represent sperm motility, forward progressing sperm. X bar represent;Start – starting sperm motility without intervention; media+ antibiotic – sperm motility after addition of media and antibiotic; h0 – represent culture before induction, at the time of induction with IPTG; h-2 represent culture after 2 hours induction; and h-4 represent culture after 4 hours of induction.<br><b>Conclusion</b>: Media containing antibiotic inhibit sperm motility by inhibiting its forward progression, thus, this data disrupt the other data. However, average of C1,C2, and C3 shows more sperm inhibition than wild type E. coli BL21 CP.";

     document.getElementById("isi").innerHTML = "<img src='https://static.igem.org/mediawiki/2015/2/24/UI_INDONESIA_GRANDDESIGN.png' width='500px'><br>We design a device for gram positive bacteria. After discussion, we chose to use B. subtilis 168and E. coli BL21as our host. B. subtilis 168 is a model for gram positive toggle switch. E. coli BL21 and B. subtilis 168 are chosen for chassis for expression of SboA.<br>For grand design we designeda toggle switch with the spermicidal protein; Subtilosin A (SboA) in the system. In the other side of the switch, we designed a suicide protein: NdoA.<br>The promoter in the system are Phyperspank(Phs) and PxylA. Phyperspank is a constitutive promoter  for gram positive when the repressor protein, LacI is not present. When present, LacI bound to Phs, inhibiting the expression of the promoter. However, with addition of lactose or IPTG, LacI would bind to IPTG and detach from the promoter, causing the promoter to express the protein downstream.<br>PxylA is another constitutive promoter for gram positive. PxylA is repressed by xylR. Xylose would bind to xylR and cause PxylA to express the protein downstream.<br>We express xylR under the effect of Phs and express LacI under the effect of PxylA. Thus, when one promoter is active, it repress the other promoter. When PxylA is active, it express SboA, the spermicidal protein. It also express LacI which inhibit the other promoter, Phs. When induced with IPTG, LacI binds to IPTG and inhibition on Phs decrease. Thus protein downstream of Phs active, which is XylR. XylR repress PxylA, thus the expression of SboA stop, and the production of NdoA started.<br>This Grand Design was the ideal design. However, we are not testing this device as a whole due to safety reason. The safety reason issue is the pathogenicity of the bacteria if we test this device as a whole.<br><br><b>SboA Expression</b><br><img src='https://static.igem.org/mediawiki/2015/7/74/UI_Indonesia_Granddesign2.png' width='200px'><br>For characterization of SboA in E. coli, we use pET9a with T7 promoter. The plasmid was transformed to E. coli BL21 CP. Culture was grown overnight. 1/10 of the culture was transferred to TB medium of 5 mL. It was shaken for 2 hours. 500μL of the culture was taken as a control before induction, the rest was induced with final concentration of 1mM IPTG. Culture of 2 hours and 4 hours after induction was taken and centrifuged. The pellet and the supernatant was taken.<br><br><b>Toggle Switch</b><br><br><img src='https://static.igem.org/mediawiki/2015/b/b6/UI_Indonesia_ToggleSwitvch.png' width='500px'><br>To characterize the toggle switch, we switch the SboA with GFP and NdoA with RFP. Single colony was streaked over LB agar with antibiotic. After 18 hours, the culture was induced with 1M IPTG and observed under UV.<br>Other characterization method involve culture of overnight colony to TB broth. After 2 hours shaking, the culture was induced with 1mM final concentration of IPTG. Another culture was induced with 1% xylose. The result was observed under UV.<br><br><b>Method</b><br>a. Sperm Motility Assay<br>Sperm Motility Assay was performed to see the engineered subtilosin A effect on human sperm<br>For motility assay, we use human spermatozoa. Ethical clearance was issued. Subject was informed and consented.<br>Sample was collected by self masturbation. It was immediately (within 1 hour) observed under light microscope. On counting sperm motility, we ask professional help from Departement of Biology Universitas Indonesia. The method was random, blind study.<br>Modified Sutyak KE, et al was used to determine the sperm motility assay. 200μL of culture was mixed with 40μL of whole semen.  The percentage of forward moving spermatozoa was documented.<br><br>b. Well Diffusion Assay<br>Well diffusion assay was performed to see Subtilosin A effectivity as bacteriocin.A 3 hour culture of M. luteus in Nutrient broth was spread in blood agar and Mueller Hinton Aga.<br><br><b>Result</b><br>a. Sperm Motility Assay<br>Decrease in sperm motility was observed after intervention. We add Media+antibiotic as a control for observing sperm death due to addition of media +antibiotic (TB+tetracycline).C1, C2, and C3 was culture 1,2, and 3 respectively. WT represent wild type BL21 CP without plasmid.<br><img src='https://static.igem.org/mediawiki/2015/f/f6/Ui_indonesia_tabel1oi.png' width='400px'><br>Sperm Motility Assay after Intervention<br><img src='https://2015.igem.org/File:Ui_indonesia_tabel2oi.png' width='400px'><br>Sperm motility compared to wild type<br><img src='https://static.igem.org/mediawiki/2015/7/7c/Ui_indonesia_tabel3oi.png' width='400px'><br><br>Graph explanation<br>Y bar represent sperm motility, forward progressing sperm. X bar represent;Start – starting sperm motility without intervention; media+ antibiotic – sperm motility after addition of media and antibiotic; h0 – represent culture before induction, at the time of induction with IPTG; h-2 represent culture after 2 hours induction; and h-4 represent culture after 4 hours of induction.<br><b>Conclusion</b>: Media containing antibiotic inhibit sperm motility by inhibiting its forward progression, thus, this data disrupt the other data. However, average of C1,C2, and C3 shows more sperm inhibition than wild type E. coli BL21 CP.";

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