Template:Team:Uppsala/JS/notebook

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ds = '<div id="dairy-text"><h1>Thursday 2013-06-27</h1> Name of participants: Marcus H, Lovisa P, Emil M, Peter C, Ken B-A, Karl H, Kristoffer L, Christoffer A, Mikael S, Jens I, Nafisa B, Stephanie H, Alona N, Viktor T, Viktor B, Nils, Alexander, Thorsten, Pontus, Magnus, Malin <h2>Ongoing constructs:</h2> L. reuteri: 1. CF48-pLR581 2. 1063-pLUL631 3. DSM 20016-pVs2 4. 100-23-noplasmid 6. DSM 20016-pLUL63TsA8 7. DSM 20016-pGFP 8. 100-23-pGT232 12. DSM 20016-noplasmid 14. DSM 20016-pLUL631(B?) L. plantarum: 5. 256-rifR-pAMβ1 10. 256-noplasmid 11. 36E-noplasmid 9. JH2-2 pAMβ1 L. lactis: 13. MG1363-pJP059 15. unknown-pGus 16. MG1363-noplasmid E.coli: 3. pSB3T5-red 4. pSB1A3-red 5. pSB4K15 7. pSB3K3-red 11. pSB4A15 14. pET13b-zifz:4cl-PBSII:STS 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT 16. pSB1C3-TAL_m 17. pSB1C3-4Cl_m 18. pSB1C3-CHS_m 19.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1 20.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1 21.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268 23.pSB1C3-CrtB 24.pSB1C3-CrtI 25.pSB1C3-CrtY 42. D5α with plasmid pSB4S15-red 43. pSB1C3-B0034-TAL 44. pSB1C3-B0034-His-TAL 45. pSB1C3-B0034-4CL 46. pSB1C3-B0034-His-4CL 47. pSB1C3-B0034-STS 48. pSB1C3-B0034-His-STS <h2>Todays work</h2> Transformation: Lb11. plantarum 36E pVS2 Lb10. plantarum 256 pLUL634 Lb12. plantarum DSM20016 pVS2 Assembly: 23.Crt.B + pEL3A15 24.Crt.I + pEL3K16 Frozen stock: Lb9. faecalis JH2-2 pAMβ1 Lb13. Lc. lactis MG1363 pJP059 clone 1 Lb15. Lc. lactis MG1363 pGus clone 1 Lb16. Lc. lactis MG1363 no plasmid clone 1 Plasmid preparation: Lb13. Lc. lactis MG1363 pJP059 clone 1 Lb15. Lc. lactis MG1363 pGus clone 1 Lb9. faecalis JH2-2 pAMβ1 Over night: 3. pSB3T5-red 4. pSB1A3-red 5. pSB4K15 7. pSB3K3-red 11. pSB4A15 Gel electrophoresis: 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT 19. (digest from previous day) 20. (“) 21. (“) PCR amplification of construct: miraculin 19.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1 20.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1 21.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268 25. pSB1C3-CrtY 43. pSB1C3-B0034-TAL 44. pSB1C3-B0034-His-TAL 45. pSB1C3-B0034-4CL 46. pSB1C3-B0034-His-4CL 47. pSB1C3-B0034-STS 48. pSB1C3-B0034-His-STS O/N screening PCR: 19.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1 20.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1 21.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268 25.pSB1C3-CrtY <h2>Results</h2> Previous day: O/N: Lb13. Lc. lactis MG1363 pJP059 clone 2: Visual pellet, foggy medium; nc ok! * Lb15. Lc. lactis MG1363 pGus clone 2: Visual pellet, foggy medium; nc ok! * Lb16. Lc. lactis MG1363 no plasmid clone 2: Visual pellet, foggy medium; nc ok!* Lb13. Lc. lactis MG1363 pJP059 clone 1: Visual pellet, slightly foggy medium; nc ok! ** Lb15. Lc. lactis MG1363 pGus clone 1: Visual pellet, slightly foggy medium; nc ok! ** Lb16. Lc. lactis MG1363 no plasmid clone 1: Visual pellet, slightly foggy medium; nc ok! ** * Optimized conditions = 10µg/ml antibiotics, fill almost up the falcon tube with M17-broth to get rid of the air, let grow in 30o C, make sure that the bacteria pellet is dissolved. → Let grow for one more day ** → Frozen stock and plasmid prep. Transformation: 42. pSB4S15-red: Many small white colonies, contamination with cocci? → Discard, get clones from Erik G. Streaking of strains: Lb13. Lc. lactis MG1363 pJP059: Some growth. Lb15. Lc. lactis MG1363 pGus: Hard to see without opening lock. Lb16. Lc. lactis MG1363 no plasmid: Hard to see without opening lock. → let grow one more day. Today: Plasmid preparation: Lb9. faecalis JH2-2 pAMβ1 clone 1: 10.1 ng/µl Lb9. faecalis JH2-2 pAMβ1 clone 2: 24.7 ng/µl Lb13. Lc. lactis MG1363-pJP059 clone 1: 4.9 ng/µl (run 1) Lb13. Lc. lactis MG1363-pJP059 clone 1: 6.0 ng/µl (run 2) Lb13. Lc. lactis MG1363-pJP059 clone 1: 6.1 ng/µl (run 3) Lb15. Lc. lactis MG1363 pGus clone 1: 61 ng/µl (run 1) → Measure again tomorrow Gel electrophoresis: Miraculin: fail, problems with primers, probably due to problematic secondary structure. 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT: failed, ladder too, probably problem with gel. Image 1 (2013.06.27) 19. (digest from previous day): Success 20. (“): Success 21. (“): Success 19. (PCR): Fail -> redo with lower TM 20. (“): Fail -> redo with lower TM 21. (“): Fail -> redo with lower TM 25. pSB1C3-CrtY: Good results <h2>Other experiments</h2> Preparation of THMS buffer</div>'

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ds = '<div id="dairy-text"><h2>Wednesday 2013-06-26</h2> Name of participants: Christoffer A, Stephanie H, Alona N, Anders E, Mikael S, Anton B, Viktor B, Marcus H, Lovisa P, Emil M, Ken B-A, Karl H, Theodor L, Magnus B., Malin B., Thorsteinn O., Nils A., Alexander W., Niclas S., Pontus D., Christoffer F. <h2>Ongoing constructs:</h2> L. reuteri: 1. CF48-pLR581 2. 1063-pLUL631 3. DSM 20016-pVs2 4. 100-23-noplasmid 6. DSM 20016-pLUL63TsA8 7. DSM 20016-pGFP 8. 100-23-pGT232 12. DSM 20016-noplasmid 14. DSM 20016-pLUL631(B?) L. plantarum: 5. 256-rifR-pAMβ1 10. 256-noplasmid 11. 36E-noplasmid E. faecalis: 9. JH2-2 pAMβ1 L. lactis 13. MG1363-pJP059 15. unknown-pGus 16. MG1363-noplasmid E.coli: 14. pET13b-zifz:4cl-PBSII:STS 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT 16. pSB1C3-TAL_m 17. pSB1C3-4Cl_m 18. pSB1C3-CHS_m 25.pSB1C3-CrtY 19.psB4C5-CrtE~O-Z / w. linker, ZF & RBS 20.psB4C5-CrtE~Z / w. linker, ZF & RBS 21.psB4C5-CrtE~I / w. linker, ZF & RBS 42. D5α with plasmid pSB4S15-red <h2>Todays work</h2> Competent cells preparation: Lb11. plantarum 36E no plasmid Lb10. plantarum 256 no plasmid Lb12. reuteri DSM 20016 no plasmid Transformation: plantarum 36E pAMB1 plantarum 256 pAMB1 Lb3. reuteri DSM 20016 pVS2 25.pSB1C3-CrtY 42. pSB4S15-red O/N: Lb5.2 plantarum 256 pAMβ1* Lb8.2 reuteri 100-23 pGT232* Lb13. Lc. lactis MG1363 pJP059** Lb15. Lc. lactis MG1363 pGus** Lb16. Lc. lactis MG1363 no plasmid** *Two tubes of 8.2 were prepared. It has grown rather poorly to not at all before. If it continues to grow poorly we may have to redo O/N from a plate rather than from our frozen stock. ** Optimized conditions → 10µg/ml antibiotics, fill almost up the falcon tube with M17-broth to get rid of the air, let grow in 30o C, make sure that the bacteria pellet is dissolved. Digestion: 19.1.psB4C5-CrtE~O-Z / w. linker, ZF & RBS 19.2psB4C5-CrtE~O-Z / w. linker, ZF & RBS 20.1.psB4C5-CrtE~Z / w. linker, ZF & RBS 20.2.psB4C5-CrtE~Z / w. linker, ZF & RBS 21.1.psB4C5-CrtE~I / w. linker, ZF & RBS 21.2.psB4C5-CrtE~I / w. linker, ZF & RBS Gel electrophoresis: 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT PCR amplification of construct: 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT (third try, using same concentration of template as second try but lower annealing temperature ) 19.1.psB4C5-CrtE~O-Z / w. linker, ZF & RBS 19.2psB4C5-CrtE~O-Z / w. linker, ZF & RBS 20.1.psB4C5-CrtE~Z / w. linker, ZF & RBS 20.2.psB4C5-CrtE~Z / w. linker, ZF & RBS 21.1.psB4C5-CrtE~I / w. linker, ZF & RBS 21.2.psB4C5-CrtE~I / w. linker, ZF & RBS <h2>Results</h2> Previous day: O/N: Lb13. Lc. lactis MG1363 pJP059: Growing, visual pellet.* Lb15. Lc. lactis MG1363 pGus: Growing, visual pellet.* Lb16. Lc. lactis MG1363 no plasmid: Growing, visual pellet.* *Weak growth in the morning, better in the afternoon. → Let grow for one more day + new O/N with optimized conditions. Streaking of strains: Lb13. Lc. lactis MG1363 pJP059: No visual growth → Let grow for one more day. Lb15. Lc. lactis MG1363 pGus: No visual growth → --”-- Lb16. Lc. lactis MG1363 no plasmid: No visual growth → --”— Today: Gel electrophoresis: 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT: Failed, possibly nonspecific primer binding, not enough template DNA. Image 1 (2013.06.26) Transformation: 25.pSB1C3-CrtY: failed D5alpha competent cells were competent <h2>Other experiments</h2> Preparation of cold electroporation buffer for competent cells Preparation of 50% glycerol stock: 500 ml 50% glycerol stock was prepared from 86-88% glycerol.</div>'

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ds = '<div id="dairy-text"><h1>Tuesday 2013-06-25</h1> Name of participants: Marcus H, Lovisa P, Emil M, Ken B-A, Karl H, Peter C, Theodor L, Christoffer A, Nafisa B, Stephanie H, Alona N, Anders E, Viktor T, Viktor B, Mikael S, Anton B <h2>Ongoing constructs:</h2> L. reuteri: 1. CF48-pLR581 2. 1063-pLUL631 3. DSM 20016-pVs2 4. 100-23-noplasmid 6. DSM 20016-pLUL63TsA8 7. DSM 20016-pGFP 8. 100-23-pGT232 12. DSM 20016-noplasmid 14. DSM 20016-pLUL631(B?) L. plantarum: 5. 256-rifR-pAMβ1 10. 256-noplasmid 11. 36E-noplasmid E. faecalis: 9. JH2-2 pAMβ1 L. lactis: 13. MG1363-pJP059 15. unknown-pGus 16. MG1363-noplasmid 14. pET13b-zifz:4cl-PBSII:STS 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT 16. pSB1C3-TAL_m 17. pSB1C3-4Cl_m 18. pSB1C3-CHS_m <h2>Todays work</h2> Gel electrophoresis: 14. pET13b-zifz:4cl-PBSII:STS 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT 16. pSB1C3-TAL_m 17. pSB1C3-4Cl_m 18. pSB1C3-CHS_m PCR amplification of construct: 15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT (second try, using smaller concentration of template and higher annealing temperature ) Lb13. Lc. lactis MG1363 pJP059 Competent cells preparation: Lb11. plantarum 36E no plasmid Lb10. plantarum 256 noplasmid Lb12. reuteri DSM 20016 no plasmid Plasmid preparation: Lb6.1 reuteri DSM 20016 pLUL634TsA8 Lb6.2 reuteri DSM 20016 pLUL634TsA8 Lb6.3 reuteri DSM 20016 pLUL634TsA8 Lb6.4 reuteri DSM 20016 pLUL634TsA8 * Lb7.2 reuteri DSM 20016 pGFP Lb7.3 reuteri DSM 20016 pGFP Lb7.4 reuteri DSM 20016 pGFP * Lb14.1 reuteri DSM 20016 pLUL631 Lb14.2 reuteri DSM 20016 pLUL631 Lb14.3 reuteri DSM 20016 pLUL631 Lb14.4 reuteri DSM 20016 pLUL631 * * 6.4, 7.4 and 14.4 were treated in a different way than the regular plasmid prep protocol for L. reuteri. Instead of treating them with LYSIS-buffer and lysozyme they were treated with resuspension solution and lysis solution from the plasmid prep kit and allowed to lyse for ~30 minutes. The rest were treated as usual, but with 1 ml of 10 mg/ml lysozyme instead of 2 ml. O/N: Lb13. Lc. lactis MG1363 pJP059 Lb15. Lc. lactis MG1363 pGus Lb16. Lc. lactis MG1363 no plasmid Streaking of strains: Lb13. Lc. lactis MG1363 pJP059 Lb15. Lc. lactis MG1363 pGus Lb16. Lc. lactis MG1363 no plasmid PCR amplification: Lb13. Lc. lactis MG1363 pJP059* *Another attempt at colony PCR with different concentrations of lysozyme and buffer. <h2>Results</h2> Gel electrophoresis: 15. failed. Possibly nonspecific primer binding, too much template DNA. Image 1 (2013.06.25) Lb13. Lc. lactis MG1363 pJP059: Only short bands, probably primer dimers because lysing failed. → Try again with a plasmid prep instead of culture. Competant cells: Wrong volume was added. → Try again tomorrow with the right volume. Plasmid preparation: * Lb6.1 reuteri DSM 20016 pLUL634 TsA8: 203.7 ng/µl Lb6.2 reuteri DSM 20016 pLUL634 TsA8: 203.2 ng/µl Lb6.3 reuteri DSM 20016 pLUL634 TsA8: 154.8 ng/µl Lb6.4 reuteri DSM 20016 pLUL634 TsA8: 39.5 ng/µl Lb7.2 reuteri DSM 20016 pGFP: 100.5 ng/µl Lb7.3 reuteri DSM 20016 pGFP: 141.2 ng/µl Lb7.4 reuteri DSM 20016 pGFP: 8.7 ng/µl Lb14.1 reuteri DSM 20016 pLUL631: 162.7 ng/µl Lb14.2 reuteri DSM 20016 pLUL631: 85.2 ng/µl Lb14.3 reuteri DSM 20016 pLUL631: 49.8 ng/µl Lb14.4 reuteri DSM 20016 pLUL631: 7.7 ng/µl *The alternative way of lysing the cells proved to be quite ineffective so we will continue to use our standard protocol. The earlier plasmid preparations of the above “constructs”, that had far poorer concentrations, were replaced with these better ones. <h2>Other experiments</h2> Preparation of MRS broth Making plates for Lc. lactis cultivation (from M17-broth) Preparation of homemade yogurt from dried bacteria and from commercial yogurt with active bacteria culture</div>'

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 	ds = '<div id="dairy-text"><h1>Thursday 2013-06-27</h1><br><b>Name of participants: </b>Marcus H, Lovisa P, Emil M, Peter C, Ken B-A, Karl H, Kristoffer L, Christoffer A, Mikael S, Jens I, Nafisa B, Stephanie H, Alona N, Viktor T, Viktor B, Nils, Alexander, Thorsten, Pontus, Magnus, Malin<br><br><h2>Ongoing constructs:</h2><br><b>L. reuteri: </b><br>1. CF48-pLR581<br>2. 1063-pLUL631<br>3. DSM 20016-pVs2<br>4. 100-23-noplasmid<br>6. DSM 20016-pLUL63TsA8<br>7. DSM 20016-pGFP<br>8. 100-23-pGT232<br>12. DSM 20016-noplasmid<br>14. DSM 20016-pLUL631(B?)<br><br><b>L. plantarum:</b><br>5. 256-rifR-pAMβ1<br>10. 256-noplasmid<br>11. 36E-noplasmid<br>9. JH2-2 pAMβ1<br><br><b>L. lactis:</b><br>13. MG1363-pJP059<br>15. unknown-pGus<br>16. MG1363-noplasmid<br><br><b>E.coli:</b><br>3. pSB3T5-red<br>4. pSB1A3-red<br>5. pSB4K15<br>7. pSB3K3-red<br>11. pSB4A15<br>14. pET13b-zifz:4cl-PBSII:STS<br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT<br>16. pSB1C3-TAL_m<br>17. pSB1C3-4Cl_m<br>18. pSB1C3-CHS_m<br>19.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1<br>20.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>21.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268<br>23.pSB1C3-CrtB<br>24.pSB1C3-CrtI<br>25.pSB1C3-CrtY<br>42. D5α with plasmid pSB4S15-red <br>43. pSB1C3-B0034-TAL<br>44. pSB1C3-B0034-His-TAL<br>45. pSB1C3-B0034-4CL<br>46. pSB1C3-B0034-His-4CL<br>47. pSB1C3-B0034-STS<br>48. pSB1C3-B0034-His-STS<br><br><h2>Todays work</h2><br><b>Transformation:</b><br>Lb11. plantarum 36E pVS2<br>Lb10. plantarum 256 pLUL634<br>Lb12. plantarum DSM20016 pVS2<br><br><b>Assembly:</b><br>23.Crt.B + pEL3A15<br>24.Crt.I + pEL3K16<br><br><b>Frozen stock: </b><br>Lb9. faecalis JH2-2 pAMβ1<br>Lb13. Lc. lactis MG1363 pJP059 clone 1<br>Lb15. Lc. lactis MG1363 pGus clone 1<br>Lb16. Lc. lactis MG1363 no plasmid clone 1<br><br><b>Plasmid preparation: </b><br>Lb13. Lc. lactis MG1363 pJP059 clone 1 <br>Lb15. Lc. lactis MG1363 pGus clone 1<br>Lb9. faecalis JH2-2 pAMβ1<br><br><b>Over night:</b><br>3. pSB3T5-red<br>4. pSB1A3-red<br>5. pSB4K15<br>7. pSB3K3-red<br>11. pSB4A15<br><br><b>Gel electrophoresis:</b><br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT<br>19. (digest from previous day)<br>20. (“)<br>21. (“)<br><br><b>PCR amplification of construct:</b><br>miraculin<br>19.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1<br>20.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>21.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268<br>25. pSB1C3-CrtY<br>43. pSB1C3-B0034-TAL<br>44. pSB1C3-B0034-His-TAL<br>45. pSB1C3-B0034-4CL<br>46. pSB1C3-B0034-His-4CL<br>47. pSB1C3-B0034-STS<br>48. pSB1C3-B0034-His-STS<br><br><b>O/N screening PCR:</b><br>19.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-HivC-Linker-CrtO-B0034-CrtZ-Linker-Gli1<br>20.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268-B0034-PBSII-Linker-CrtY-B0034-CrtZ-Linker-Gli1<br>21.pSB4C5-pBAD/AraC-B0034-Jazz-Linker-CrtE-B0034-Blues-Linker-CrtB-B0034-CrtI-Linker-Zif268<br>25.pSB1C3-CrtY<br><br><h2>Results</h2><br><b>Previous day:</b><br><b>O/N:</b> <br>Lb13. Lc. lactis MG1363 pJP059 clone 2: Visual pellet, foggy medium; nc ok! * <br>Lb15. Lc. lactis MG1363 pGus clone 2: Visual pellet, foggy medium; nc ok! *<br>Lb16. Lc. lactis MG1363 no plasmid clone 2: Visual pellet, foggy medium; nc ok!*<br>Lb13. Lc. lactis MG1363 pJP059 clone 1: Visual pellet, slightly foggy medium; nc ok! **<br>Lb15. Lc. lactis MG1363 pGus clone 1: Visual pellet, slightly foggy medium; nc ok! **<br>Lb16. Lc. lactis MG1363 no plasmid clone 1: Visual pellet, slightly foggy medium; nc ok! **<br>* Optimized conditions = 10µg/ml antibiotics, fill almost up the falcon tube with M17-broth to get rid of the air, let grow in 30o C, make sure that the bacteria pellet is dissolved. → Let grow for one more day <br>** → Frozen stock and plasmid prep.<br><br><b>Transformation: </b><br>42. pSB4S15-red: Many small white colonies, contamination with cocci?<br>→ Discard, get clones from Erik G.<br><br><b>Streaking of strains:</b><br>Lb13. Lc. lactis MG1363 pJP059: Some growth.<br>Lb15. Lc. lactis MG1363 pGus: Hard to see without opening lock.<br>Lb16. Lc. lactis MG1363 no plasmid: Hard to see without opening lock.<br>→ let grow one more day.<br><br><b>Today:</b><br><b>Plasmid preparation: </b><br>Lb9. faecalis JH2-2 pAMβ1 clone 1: 10.1 ng/µl<br>Lb9. faecalis JH2-2 pAMβ1 clone 2: 24.7 ng/µl<br>Lb13. Lc. lactis  MG1363-pJP059 clone 1: 4.9 ng/µl (run 1)<br>Lb13. Lc. lactis MG1363-pJP059 clone 1: 6.0 ng/µl (run 2)<br>Lb13. Lc. lactis MG1363-pJP059 clone 1: 6.1 ng/µl (run 3)<br>Lb15. Lc. lactis MG1363 pGus clone 1: 61 ng/µl (run 1)<br>→ Measure again tomorrow<br><br><b>Gel electrophoresis:</b><br>Miraculin: fail, problems with primers, probably due to problematic secondary structure.<br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT: failed, ladder too, probably problem with gel. Image 1 (2013.06.27)<br>19. (digest from previous day): Success<br>20. (“): Success<br>21. (“): Success<br>19. (PCR): Fail -> redo with lower TM<br>20. (“): Fail -> redo with lower TM<br>21. (“): Fail -> redo with lower TM<br>25. pSB1C3-CrtY: Good results<br><br><h2>Other experiments</h2><br><b>Preparation of THMS buffer</b></div>'
   }
-else if(id == 'd2013625')
+else if(id == 'd2013626')
   {
 	ds = '<div id="dairy-text"><h2>Wednesday 2013-06-26</h2><br><b>Name of participants: </b>Christoffer A, Stephanie H, Alona N, Anders E, Mikael S, Anton B, Viktor B, Marcus H, Lovisa P, Emil M, Ken B-A, Karl H, Theodor L, Magnus B., Malin B., Thorsteinn O., Nils A., Alexander W., Niclas S., Pontus D., Christoffer F.<br><br><h2>Ongoing constructs:</h2><br><b>L. reuteri: </b><br>1. CF48-pLR581<br>2. 1063-pLUL631<br>3. DSM 20016-pVs2<br>4. 100-23-noplasmid<br>6. DSM 20016-pLUL63TsA8<br>7. DSM 20016-pGFP<br>8. 100-23-pGT232<br>12. DSM 20016-noplasmid<br>14. DSM 20016-pLUL631(B?)<br><br><b>L. plantarum:</b><br>5. 256-rifR-pAMβ1<br>10. 256-noplasmid<br>11. 36E-noplasmid<br><br><b>E. faecalis:</b><br>9. JH2-2 pAMβ1<br><br><b>L. lactis</b><br>13. MG1363-pJP059<br>15. unknown-pGus<br>16. MG1363-noplasmid<br><br><b>E.coli:</b><br>14. pET13b-zifz:4cl-PBSII:STS<br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT<br>16. pSB1C3-TAL_m<br>17. pSB1C3-4Cl_m<br>18. pSB1C3-CHS_m<br>25.pSB1C3-CrtY<br>19.psB4C5-CrtE~O-Z / w. linker, ZF & RBS<br>20.psB4C5-CrtE~Z / w. linker, ZF & RBS<br>21.psB4C5-CrtE~I / w. linker, ZF & RBS<br>42. D5α with plasmid pSB4S15-red<br><br><h2>Todays work</h2><br><b>Competent cells preparation:</b><br>Lb11. plantarum 36E no plasmid<br>Lb10. plantarum 256 no plasmid<br>Lb12. reuteri DSM 20016 no plasmid<br><br><b>Transformation:</b><br>plantarum 36E pAMB1<br>plantarum 256 pAMB1<br>Lb3. reuteri DSM 20016 pVS2<br>25.pSB1C3-CrtY<br>42. pSB4S15-red <br><br><b>O/N:</b><br>Lb5.2 plantarum 256 pAMβ1*<br>Lb8.2 reuteri 100-23 pGT232*<br>Lb13. Lc. lactis MG1363 pJP059**<br>Lb15. Lc. lactis MG1363 pGus**<br>Lb16. Lc. lactis MG1363 no plasmid**<br>*Two tubes of 8.2 were prepared. It has grown rather poorly to not at all before. If it continues to grow poorly we may have to redo O/N from a plate rather than from our frozen stock.<br>** Optimized conditions → 10µg/ml antibiotics, fill almost up the falcon tube with M17-broth to get rid of the air, let grow in 30o C, make sure that the bacteria pellet is dissolved.  <br><br><b>Digestion:</b><br>19.1.psB4C5-CrtE~O-Z / w. linker, ZF & RBS<br>19.2psB4C5-CrtE~O-Z / w. linker, ZF & RBS<br>20.1.psB4C5-CrtE~Z / w. linker, ZF & RBS<br>20.2.psB4C5-CrtE~Z / w. linker, ZF & RBS<br>21.1.psB4C5-CrtE~I / w. linker, ZF & RBS<br>21.2.psB4C5-CrtE~I / w. linker, ZF & RBS<br><br><b>Gel electrophoresis:</b><br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT<br><br><b>PCR amplification of construct:</b><br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT (third try, using same concentration of template as second try but lower annealing temperature )<br>19.1.psB4C5-CrtE~O-Z / w. linker, ZF & RBS<br>19.2psB4C5-CrtE~O-Z / w. linker, ZF & RBS<br>20.1.psB4C5-CrtE~Z / w. linker, ZF & RBS<br>20.2.psB4C5-CrtE~Z / w. linker, ZF & RBS<br>21.1.psB4C5-CrtE~I / w. linker, ZF & RBS<br>21.2.psB4C5-CrtE~I / w. linker, ZF & RBS<br><br><br><h2>Results</h2><br><b>Previous day:</b><br><b>O/N:</b> <br>Lb13. Lc. lactis MG1363 pJP059: Growing, visual pellet.*<br>Lb15. Lc. lactis MG1363 pGus: Growing, visual pellet.*<br>Lb16. Lc. lactis MG1363 no plasmid: Growing, visual pellet.*<br>*Weak growth in the morning, better in the afternoon. → Let grow for one more day + new O/N with optimized conditions.<br><br><b>Streaking of strains: </b><br>Lb13. Lc. lactis MG1363 pJP059: No visual growth → Let grow for one more day.<br>Lb15. Lc. lactis MG1363 pGus: No visual growth →		--”--<br>Lb16. Lc. lactis MG1363 no plasmid: No visual growth → 	--”—<br><br><b>Today:</b><br><b>Gel electrophoresis:</b><br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT: Failed, possibly nonspecific primer binding, not enough template DNA. Image 1 (2013.06.26)<br><br><b>Transformation:</b><br>25.pSB1C3-CrtY: failed<br>D5alpha competent cells were competent<br><br><br><h2>Other experiments</h2><br><b>Preparation of cold electroporation buffer for competent cells</b><br><br><b>Preparation of 50% glycerol stock: </b><br>500 ml 50% glycerol stock was prepared from 86-88% glycerol.</div>'
   }
+ else if(id == 'd2013625')
   {
 	ds = '<div id="dairy-text"><h1>Tuesday 2013-06-25</h1><br><br><b>Name of participants:</b> Marcus H, Lovisa P, Emil M, Ken B-A, Karl H, Peter C, Theodor L, Christoffer A, Nafisa B, Stephanie H, Alona N, Anders E, Viktor T, Viktor B, Mikael S, Anton B<br><br><h2>Ongoing constructs:</h2><br><b>L. reuteri: </b><br>1. CF48-pLR581<br>2. 1063-pLUL631<br>3. DSM 20016-pVs2<br>4. 100-23-noplasmid<br>6. DSM 20016-pLUL63TsA8<br>7. DSM 20016-pGFP<br>8. 100-23-pGT232<br>12. DSM 20016-noplasmid<br>14. DSM 20016-pLUL631(B?)<br><br><b>L. plantarum:</b><br>5. 256-rifR-pAMβ1<br>10. 256-noplasmid<br>11. 36E-noplasmid<br><br><b>E. faecalis:</b><br>9. JH2-2 pAMβ1<br><br><b>L. lactis:</b><br>13. MG1363-pJP059<br>15. unknown-pGus<br>16. MG1363-noplasmid<br>14. pET13b-zifz:4cl-PBSII:STS<br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT<br>16. pSB1C3-TAL_m<br>17. pSB1C3-4Cl_m<br>18. pSB1C3-CHS_m<br><br><h2>Todays work</h2><br><b>Gel electrophoresis:</b><br>14. pET13b-zifz:4cl-PBSII:STS<br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT<br>16. pSB1C3-TAL_m<br>17. pSB1C3-4Cl_m<br>18. pSB1C3-CHS_m<br><br><b>PCR amplification of construct:</b><br>15. pSB4K5-RBS-TAL-Linker-Zif268-RBS-4cl-Linker-PBSII-STS-OMT (second try, using smaller concentration of template and higher annealing temperature )<br>Lb13. Lc. lactis MG1363 pJP059<br><br><b>Competent cells preparation: </b><br>Lb11. plantarum 36E no plasmid<br>Lb10. plantarum 256 noplasmid<br>Lb12. reuteri DSM 20016 no plasmid<br><br><b>Plasmid preparation: </b><br>Lb6.1 reuteri DSM 20016 pLUL634TsA8<br>Lb6.2 reuteri DSM 20016 pLUL634TsA8<br>Lb6.3 reuteri DSM 20016 pLUL634TsA8<br>Lb6.4 reuteri DSM 20016 pLUL634TsA8 *<br>Lb7.2 reuteri DSM 20016 pGFP<br>Lb7.3 reuteri DSM 20016 pGFP<br>Lb7.4 reuteri DSM 20016 pGFP *<br>Lb14.1 reuteri DSM 20016 pLUL631<br>Lb14.2 reuteri DSM 20016 pLUL631<br>Lb14.3 reuteri DSM 20016 pLUL631<br>Lb14.4 reuteri DSM 20016 pLUL631 *<br>* 6.4, 7.4 and 14.4 were treated in a different way than the regular plasmid prep protocol for L. reuteri. Instead of treating them with LYSIS-buffer and lysozyme they were treated with resuspension solution and lysis solution from the plasmid prep kit and allowed to lyse for ~30 minutes. The rest were treated as usual, but with 1 ml of 10 mg/ml lysozyme instead of 2 ml.<br><br><b>O/N:</b> <br>Lb13. Lc. lactis MG1363 pJP059<br>Lb15. Lc. lactis MG1363 pGus<br>Lb16. Lc. lactis MG1363 no plasmid<br><br><b>Streaking of strains:</b><br>Lb13. Lc. lactis MG1363 pJP059<br>Lb15. Lc. lactis MG1363 pGus<br>Lb16. Lc. lactis MG1363 no plasmid<br><br><b>PCR amplification: </b><br>Lb13. Lc. lactis MG1363 pJP059*<br>*Another attempt at colony PCR with different concentrations of lysozyme and buffer.<br><br><h2>Results</h2><br><b>Gel electrophoresis:</b><br>15. failed. Possibly nonspecific primer binding, too much template DNA. Image 1 (2013.06.25)<br>Lb13. Lc. lactis MG1363 pJP059: Only short bands, probably primer dimers because lysing failed. → Try again with a plasmid prep instead of culture.<br><br><b>Competant cells:</b><br>Wrong volume was added. → Try again tomorrow with the right volume.<br><br><b>Plasmid preparation: *</b><br>Lb6.1 reuteri DSM 20016 pLUL634 TsA8:	203.7 ng/µl<br>Lb6.2 reuteri DSM 20016 pLUL634 TsA8:	203.2 ng/µl<br>Lb6.3 reuteri DSM 20016 pLUL634 TsA8:	154.8 ng/µl<br>Lb6.4 reuteri DSM 20016 pLUL634 TsA8:	39.5 ng/µl<br>Lb7.2 reuteri DSM 20016 pGFP:		100.5 ng/µl<br>Lb7.3 reuteri DSM 20016 pGFP:		141.2 ng/µl<br>Lb7.4 reuteri DSM 20016 pGFP:		8.7 ng/µl<br>Lb14.1 reuteri DSM 20016 pLUL631:	162.7 ng/µl<br>Lb14.2 reuteri DSM 20016 pLUL631:	85.2 ng/µl<br>Lb14.3 reuteri DSM 20016 pLUL631:	49.8 ng/µl<br>Lb14.4 reuteri DSM 20016 pLUL631:	7.7 ng/µl<br>*The alternative way of lysing the cells proved to be quite ineffective so we will continue to use our standard protocol. The earlier plasmid preparations of the above “constructs”, that had far poorer concentrations, were replaced with these better ones.<br><br><h2>Other experiments</h2><br><b>Preparation of MRS broth </b><br><br><b>Making plates for Lc. lactis cultivation (from M17-broth)</b><br><br><b>Preparation of homemade yogurt from dried bacteria and from commercial yogurt with active bacteria culture</b></div>'

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