|
|
(12 intermediate revisions not shown) |
Line 23: |
Line 23: |
| | | |
| <div id="nav12d"><a href="https://2009.igem.org/Team:Queens/Project"><img src="https://static.igem.org/mediawiki/2009/2/21/QueensNavProject.png"></a></div> | | <div id="nav12d"><a href="https://2009.igem.org/Team:Queens/Project"><img src="https://static.igem.org/mediawiki/2009/2/21/QueensNavProject.png"></a></div> |
| + | |
| + | <div id="nav17d"><a href="https://2009.igem.org/Team:Queens/Results"><img src="https://static.igem.org/mediawiki/2009/3/37/QueensNavResults.png"></a></div> |
| | | |
| <div id="nav13d"><a href="https://2009.igem.org/Team:Queens/Parts"><img src="https://static.igem.org/mediawiki/2009/2/29/QueensNavParts.png"></a></div> | | <div id="nav13d"><a href="https://2009.igem.org/Team:Queens/Parts"><img src="https://static.igem.org/mediawiki/2009/2/29/QueensNavParts.png"></a></div> |
Line 28: |
Line 30: |
| <div id="nav14d"><a href="https://2009.igem.org/Team:Queens/Notebook"><img src="https://static.igem.org/mediawiki/2009/d/d4/QueensNavNotebook.png"></a></div> | | <div id="nav14d"><a href="https://2009.igem.org/Team:Queens/Notebook"><img src="https://static.igem.org/mediawiki/2009/d/d4/QueensNavNotebook.png"></a></div> |
| | | |
- | <div id="nav15d"><a href="https://2009.igem.org/Team:Queens/Modelling"><img src="https://static.igem.org/mediawiki/2009/7/75/QueensNavModelling.png"></a></div> | + | <div id="nav15d"><a href="https://2009.igem.org/Team:Queens/Protocols"><img src="https://static.igem.org/mediawiki/2009/3/3d/QueensNavProtocols.png"></a></div> |
| | | |
| <div id="nav16d"><a href="https://2009.igem.org/Team:Queens/Sponsors"><img src="https://static.igem.org/mediawiki/2009/a/a0/QueensNavSponsors.png"></a></div> | | <div id="nav16d"><a href="https://2009.igem.org/Team:Queens/Sponsors"><img src="https://static.igem.org/mediawiki/2009/a/a0/QueensNavSponsors.png"></a></div> |
Line 35: |
Line 37: |
| | | |
| <table style="background-color:#922334; position:relative; overflow:auto; left:200px; top:-215px; width:750px"> | | <table style="background-color:#922334; position:relative; overflow:auto; left:200px; top:-215px; width:750px"> |
- | <tr>
| |
- | <td align="left"><p style="font-size:175%;font-family:corbel;color:#172C4E;font-weight:bold"><i>Menu<i>
| |
- | </p></td>
| |
- | </tr>
| |
| | | |
- | <tr>
| |
- | <td align="left">
| |
- | <a href="#Lab1"><p style="font-size:120%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a>
| |
- | <a href="#Lab2"><p style="font-size:120%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a>
| |
- | <a href="#Tech"><p style="font-size:120%;font-family:corbel;color:#ECB528;font-weight:bold">Tech Support and Laboratory Three: Parthiv, Chris P., Chris Y.</p></a>
| |
- | </td>
| |
- | </tr>
| |
- |
| |
- | <tr>
| |
- | <td>
| |
| <br/> | | <br/> |
- | <br/>
| |
- | </td>
| |
- | </tr>
| |
| | | |
| <tr> | | <tr> |
| <td align="left"> | | <td align="left"> |
- | <a name="Lab1"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a>
| + | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E;"> |
- | <br/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E;">16/06/2009</p> | + | |
| <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> | | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528"> |
- | -Lab Meeting
| + | Please note that Lab Journal's are in PDF Format. |
- | -Finished the sequence for the surface expression construct
| + | Please click on the Lab Journal you wish to view. |
- | -Finalized to-do list
| + | </pre></p></td> |
- | -Mini-prepped the following:
| + | </tr> |
- | -Linker K157013 <i>Plate 3, Well 3G, plasmid Bba_K157000(A)</i>
| + | |
- | -TEV Protease
| + | |
- | -I712078 (C-Terminus) <i>Plate 2, Well 14M, plasmid J70003 (A)</i>
| + | |
- | -I712077 (N-Terminus) <i>Plate 2, Well 14K, plasmid J70003 (A)</i>
| + | |
- | -pLux
| + | |
- | -R0062 (not leaky) <i>Plate 1, Well 6O, plasmid pSB1A2 (A)</i>
| + | |
- | -R1062 (median strength in the absence of luxR/HSL)
| + | |
- | <i>Plate 1, Well 8G, plasmid pSB1A2 (A)</i>
| + | |
- | -HO-pcyA
| + | |
- | -K098010 <i>Plate 3, Well 11N, plasmid pSB3k3 (K)</i>
| + | |
- | -Terminator
| + | |
- | -B0015 <i>Plate 1, Well 23L, plasmid pSB1AK3 (AK)</i>
| + | |
- | -RFP
| + | |
- | -E1010 <i>Plate 1, Well 18F, plasmid pSB2K3 (K)</i>
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">17/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Transformed the following parts into Top10
| + | |
- | 1. K157013 <i>Plate 3, Well 3G, K157000, Res:A</i>
| + | |
- | 2. I712078 <i>Plate 2, well 14M, J70003, Res:A</i>
| + | |
- | 3. I712077 <i>Plate 2, Well 14K, J70003, Res:A</i>
| + | |
- | 4. R0062 <i>Plate 1, Well 6O, pSB1A2, Res:A</i>
| + | |
- | 5. R1062 <i>Plate 1, Well 8G, pSB1A2, Res:A</i>
| + | |
- | 6. K098910 <i>Plate 3, Well 11N, pSB3K3, Res:A</i>
| + | |
- | 7. B0015 <i>Plate 1, Well 23L, pSB1AK3, Res:AK</i>
| + | |
- | 8. E1010 <i>Plate 2, Well 18F, pSB2K3, Res:K</i>
| + | |
- | -Left in 37C for four hours and left on lab bench at room temperature overnight
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">18/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -No colonies were observered on the plates
| + | |
- | -Left the plates in 37C for four hours
| + | |
- | -Picked one colony from each plate and made overnight broth culture for glycerol stock.
| + | |
- | -K098010 plate did not grow, so it was left in 37C overnight.
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">19/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Made the glycerol stocks from the overnight broth cultures
| + | |
- | -Started broth culture for K098010
| + | |
- | -Re-transformed the E1010 in Top10
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">22/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Restarted the broth culture for K098010 because it was overgrown (already in stationary phase)
| + | |
- | -Observed no colonies on E1010 plate. Discovered that E1010 should be on Kanamycin resistance plate.
| + | |
- | | + | |
- | Things to Do:
| + | |
- | -Get out the standard plasmid backbones
| + | |
- | -High copy number assembly plasmid backbone
| + | |
- | -pSB1A3 <i>Plate 1, Well 1K</i>
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">23/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Submitted VLA construct to Mr. GENE for synthesis
| + | |
- | -Placed orders for PCR primers
| + | |
- | </pre> | + | |
- | </p> | + | |
- | <p/> | + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">24/06/2009</p> | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Transformed the following parts into Top10
| + | |
- | 9. RBS-LuxR J37033 <i>Plate 3, Well 4O, pSB1A2, Res:A</i>
| + | |
- | 10. RBS-LuxL-ter F1610 <i>Plate 2, Well 24G, pSB1AK3, Res:AK</i>
| + | |
- | 11. RBS-LuxL K081008 <i>Plate 2, Well 10L, pSB1A2, Res:A</i>
| + | |
- | 12. RBS-LuxR-ter I0462 <i>Plate 1, Well 8O, pSB1A2, Res:A</i>
| + | |
- | 13. Pconst J23119 <i>Plate 1, Well 18A, pSB1A2, Res:A</i>
| + | |
- | 14. LuxR C0062 <i>Plate 1, Well 4O, pSB1A2, Res:A</i>
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">25/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Purchased QiaMiniPrep Kit from BioBar
| + | |
- | -Picked colonies from plates and re-cultured in broth; left overnight
| + | |
- | -Ordered ITGA4 cDNA plasmid from OpenBioSystem
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">26/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Made glycerol stocks for parts transformed 23/06/2009
| + | |
- | -Designed primer for PCR out VLA cDNA plasmid and primer for pLux-RBS-HindIII
| + | |
- | -Transformed the following parts into Top10 and left the plates in 37C overnight.
| + | |
- | 15. RBS B0034 <i>Plate 1, Well 2M, pSB1A2, Res:A</i>
| + | |
- | 16. HemeC I716154 <i>Plate 1, Well 17B, pSB1A2, Res:A</i>
| + | |
- | 17. HemeD I716155 <i>Plate 1, Well 17D, pSB1A2, Res:A</i>
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- |
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">27/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Picked colonies from plates and made glycerol stocks
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">28/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Transformed gycerol stocks into broth culture
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">29/06/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Purified plasmids containing parts 1-17 from Top10 using Qiagen Spin MiniPrep Kit.
| + | |
- | -Performed Agarose Gel Electrophoresis to check the size of the parts. Bends did
| + | |
- | not migrate very far, possible due tot he fact that circular plasmids migrate very
| + | |
- | slowly. We decided to digest the plasmids with a BioBrick restriction enzyme
| + | |
- | and then run the gel again.
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">02/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Re-ran the Agarose Gel Electrophoresis to ckeck the size of the parts. It worked.
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">06/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Transformed the following parts into Top10
| + | |
- | 18. GFP E0040 <i>Plate 1, Well 14K, pSB1A2, Res:A</i>
| + | |
- | 19. GFP constr. E0840 <i>Plate 1, Well 12O, pSB1A2, Res:A</i>
| + | |
- | 20. pTet+GFP I13522 <i>Plate 2, Well 8A, pSB1A2, Res:A</i>
| + | |
- | 21. LuxR constr. K091204 <i>Plate 2, Well 8J, pSB1A2, Res:A</i>
| + | |
- | 22. LuxL+GFP J37034 <i>Plate 2, Well 7I, pSB1A2, Res:AK</i>
| + | |
- | 23. pSB1AC3 <i>Plate 1, Well 11A Res:AC</i>
| + | |
- | 24. pSB1AK3 <i>Plate 1, Well 13A Res:AK</i>
| + | |
- | 25. pSB1AT3 <i>Plate 1, Well 15A, Res:AT</i>
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">07/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Picked colonies from plates and made broth culture which was left to grow overnight
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">08/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Purified the plasmids of parts 18-25 using Qiagen Spin MiniPrep Kit.
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">09/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Ran a 1% Agarose gel electrophoresis to confirm the plasmid lengths of parts 18 to 25
| + | |
- | -Gel loading and concentrations:
| + | |
- | I. E0040 120μg/μL loaded 12μL
| + | |
- | II. pSB1AC3 120μg/μL loaded 12μL
| + | |
- | III. R0062 120μg/μL loaded 12μL
| + | |
- | IV. pSB1AT3 120μg/μL loaded 12μL
| + | |
- | V. J23119 120μg/μL loaded 12μL
| + | |
- | VI. J37034 120μg/μL loaded 12μL
| + | |
- | VII. K091204 120μg/μL loaded 12μL
| + | |
- | VIII. E0840 120μg/μL loaded 10μL
| + | |
- | IX. B0034 50μg/μL loaded 10μL
| + | |
- | X. F1610 50μg/μL loaded 10μL
| + | |
- | XI. I13522 120μg/μL loaded 10μL
| + | |
- | -Calculated the relative concentrations of the parts by comparing the bands with
| + | |
- | the ladder (The 5000bp ladder is about 120ng/μL)
| + | |
- | -Parts digested:
| + | |
- | I. R0062 Plux prefix EcoR1+Spe1
| + | |
- | II. pSB1AT3 backbone EcoR1+Pst1
| + | |
- | III. J23119 Pconst prefix EcoR1+Spe1
| + | |
- | IV. B0034 RBS suffix Xba1+Pst1
| + | |
- | V. F1610 RBS-LuxI-STOP suffix Xba1+Pst1
| + | |
- | VI. B0015 Terminator prefix EcoR1+Spe1
| + | |
- | -Restriction digestion mix recipe:
| + | |
- | -600ng of DNA
| + | |
- | -4μL restriction buffer
| + | |
- | -0.5μL EcoR1 and 0.5μL Spe1, or
| + | |
- | -0.5μL Xba1 and 0.5μL Pst1
| + | |
- | -Up to 35μL of ddH<sub>2</sub>0
| + | |
- | -Digested the parts using corresponding BioBrick restriction enzymes, and then
| + | |
- | purified parts using QiaQuick PCR Purification Kit.
| + | |
- | -Stored the purified DNA in -20C overnight
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">10/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Ran a 1% Agarose gel electrophoresis to confirm the products of restriction
| + | |
- | digestions.
| + | |
- | -The expected bands of the parts did not show up. This is probably dude to
| + | |
- | the fact that most of the parts have lengths within 100bp and the QiaQuick
| + | |
- | PCR purification kit removes DNA below 100bp length.
| + | |
- | -The miniprep plasmids of the parts are digested again with corresponding
| + | |
- | restriction enzymes for 2 hours
| + | |
- | -Enzymes are deactivated (denatured) by heating at 65C
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">13/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Ligated the parts into the following constructs using the <i>T4 Ligase Protocol</i>
| + | |
- | -P<sub>const</sub> - RBS - LuxL - 2xSTOP
| + | |
- | -P<sub>lux</sub> - RBS
| + | |
- | -P<sub>const</sub> - RBS (Heme Oxygenase)
| + | |
- | -Terminator - RBS
| + | |
- | -Made broth culture from the VLA cDNA glycerol stock
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#172C4E">14/07/2009</p>
| + | |
- | <p style="font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | <pre style="border-style:none;background-color:#922334;font-size:120%;font-family:palatino linotype;color:#ECB528">
| + | |
- | -Purified the plasmid for ITGA4 using QiaMiniPrep Spin Kit
| + | |
- | -Recultured ITGA4-containing cells in broth
| + | |
- | </pre>
| + | |
- | </p>
| + | |
- | <p/>
| + | |
- | | + | |
| | | |
| <br/> | | <br/> |
- | </td>
| |
- | </tr>
| |
| | | |
| <tr> | | <tr> |
| <td align="left"> | | <td align="left"> |
- | <a name="Lab2"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Laboratory Two: Kate, Mike</p></a> | + | <a href="https://static.igem.org/mediawiki/2009/5/5d/QueensLabNotesHarryBryantJamesBogdan.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory One: Harry, Bogdan, James, Bryant</p></a> |
- | <br/> | + | <a href="https://static.igem.org/mediawiki/2009/0/0e/QueensLabNotesKateMike.pdf"><p style="font-size:150%;font-family:corbel;color:#ECB528;font-weight:bold">Laboratory Two: Kate, Mike</p></a> |
- | | + | |
- | | + | |
- | | + | |
- | </br> | + | |
| </td> | | </td> |
| </tr> | | </tr> |
| | | |
| <tr> | | <tr> |
- | <td align="left"> | + | <td> |
- | <a name="Tech"><p style="font-size:150%;font-family:corbel;color:#172C4E;font-weight:bold">Tech Support and Laboratory Three: Parthiv, Chris P., Chris Y.</p></a>
| + | |
| <br/> | | <br/> |
- |
| |
- |
| |
- |
| |
| <br/> | | <br/> |
| </td> | | </td> |
| </tr> | | </tr> |
| + | |
| | | |
| </table> | | </table> |
Line 370: |
Line 84: |
| <br/> | | <br/> |
| <font color="#172C4E"><center> | | <font color="#172C4E"><center> |
- | Last Updated: May 11, 2009 by Fr<sub>3</sub>P</center></font> | + | Last Updated: October 20, 2009 by Fr<sub>3</sub>P</center></font> |
| | | |
| </footer> | | </footer> |
| </html> | | </html> |