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TUDelft/22 September 2008
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==3A Assembly== | ==3A Assembly== | ||
We've received our last thermopart from GeneArt and have started construction of the two remaining parts today. The parts under construction are now [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115030 K115030] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115035 K115035]. | We've received our last thermopart from GeneArt and have started construction of the two remaining parts today. The parts under construction are now [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115030 K115030] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115035 K115035]. | ||
| + | |||
| + | ==Growing induced cultures== | ||
| + | We've started low temperature (26ºC) growing of induced strains today. Cultures grown contained either pSB1AT3 + P1010, K115012, K115029, K115031, K115032 or K115034. Some had different plasmids, either pSB1AT3 or pSB1AK3, so we've grown them on Ampicillin to reduce growing conditions variation. We aimed for lysis on Wednesday (OD=0.6). However that was planned for T=20ºC, and the temperature of our water bath could not go below 26ºC. We'll lyse the cells when they are ready. | ||
| + | |||
| + | ==Protein determination== | ||
| + | To find better values to correct luciferase measurements for (instead of OD), we've measured total protein content. To make results easier to view we've made the following calculation: | ||
| + | Correct luminescence for protein content by dividing each measured luminescence by the protein content of the sample. | ||
| + | Normalize the luminescence of the thermosensitive construct K115034 for the luminescence of the control construct K115012. Results displayed in table 1 are the final outcomes for these normalized values per sample treatment. Please note that these first measurements are more important for optimalization of our protocols, not for reliable results of our constructs. | ||
| + | |||
| + | |||
| + | {| border="1" | ||
| + | |+ <b>Table 1: Normalized luciferase expression of construct K115034 under various growing/lysing conditions</b> | ||
| + | ! Growth scheme!!Lysis method!!Protein content (ug/ml)!!Normalized luciferase expression!! | ||
| + | |- | ||
| + | |Induced: 20 h @ Tr | ||
| + | |Sonification | ||
| + | |0.87 | ||
| + | |1.76 | ||
| + | |- | ||
| + | |Induced: 20 h @ 37oC | ||
| + | |Sonification | ||
| + | |1.88 | ||
| + | |0.35 | ||
| + | |- | ||
| + | |Non induced: 15 h @ 37oC; induced: 5 h @ Tr | ||
| + | |Sonification | ||
| + | |2.37 | ||
| + | |316.65 | ||
| + | |- | ||
| + | |Induced: 40 hours @ Tr | ||
| + | |Sonification | ||
| + | |2.47 | ||
| + | |0.09 | ||
| + | |- | ||
| + | |Induced: 40 hours @ Tr | ||
| + | |Promega Lysis buffer | ||
| + | |2.37 | ||
| + | |0.24 | ||
| + | |} | ||
| + | |||
| + | Much conclusions still can't be drawn on these data. There was an indication the R0080 arabinose induction might not be working properly. Either we'll have to use F2620 as promoter or just keep growth at the temperature desired. Another option might be to use the commercial pBAD vector. | ||
| + | |||
{{Template:TUDelftiGEM2008_sidebar}} | {{Template:TUDelftiGEM2008_sidebar}} | ||
Latest revision as of 12:19, 27 October 2008
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| [http://2008.igem.org/TUDelft/8_September_2008 8] | [http://2008.igem.org/TUDelft/9_September_2008 9] | [http://2008.igem.org/TUDelft/10_September_2008 10] | [http://2008.igem.org/TUDelft/11_September_2008 11] | [http://2008.igem.org/TUDelft/12_September_2008 12] | [http://2008.igem.org/wiki/index.php?title=TUDelft/13_September_2008&action=edit 13] | [http://2008.igem.org/wiki/index.php?title=TUDelft/14_September_2008&action=edit 14] |
| [http://2008.igem.org/TUDelft/15_September_2008 15] | [http://2008.igem.org/TUDelft/16_September_2008 16] | [http://2008.igem.org/TUDelft/17_September_2008 17] | [http://2008.igem.org/TUDelft/18_September_2008 18] | [http://2008.igem.org/TUDelft/19_September_2008 19] | [http://2008.igem.org/wiki/index.php?title=TUDelft/20_September_2008&action=edit 20] | [http://2008.igem.org/wiki/index.php?title=TUDelft/21_September_2008&action=edit 21] |
| [http://2008.igem.org/TUDelft/22_September_2008 22] | [http://2008.igem.org/TUDelft/23_September_2008 23] | [http://2008.igem.org/TUDelft/24_September_2008 24] | [http://2008.igem.org/TUDelft/25_September_2008 25] | [http://2008.igem.org/wiki/index.php?title=TUDelft/26_September_2008&action=edit 26] | [http://2008.igem.org/wiki/index.php?title=TUDelft/27_September_2008&action=edit 27] | [http://2008.igem.org/wiki/index.php?title=TUDelft/28_September_2008&action=edit 28] |
| [http://2008.igem.org/TUDelft/29_September_2008 29] | [http://2008.igem.org/TUDelft/30_September_2008 30] | |||||
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| [http://2008.igem.org/TUDelft/1_October_2008 1] | [http://2008.igem.org/TUDelft/2_October_2008 2] | [http://2008.igem.org/TUDelft/3_October_2008 3] | [http://2008.igem.org/wiki/index.php?title=TUDelft/4_October_2008&action=edit 4] | [http://2008.igem.org/wiki/index.php?title=TUDelft/5_October_2008&action=edit 5] | ||
| [http://2008.igem.org/TUDelft/6_October_2008 6] | [http://2008.igem.org/TUDelft/7_October_2008 7] | [http://2008.igem.org/TUDelft/8_October_2008 8] | [http://2008.igem.org/TUDelft/9_October_2008 9] | [http://2008.igem.org/TUDelft/10_October_2008 10] | [http://2008.igem.org/wiki/index.php?title=TUDelft/11_October_2008&action=edit 11] | [http://2008.igem.org/wiki/index.php?title=TUDelft/12_October_2008&action=edit 12] |
| [http://2008.igem.org/TUDelft/13_October_2008 13] | [http://2008.igem.org/TUDelft/14_October_2008 14] | [http://2008.igem.org/TUDelft/15_October_2008 15] | [http://2008.igem.org/TUDelft/16_October_2008 16] | [http://2008.igem.org/TUDelft/17_October_2008 17] | [http://2008.igem.org/wiki/index.php?title=TUDelft/18_October_2008&action=edit 18] | [http://2008.igem.org/wiki/index.php?title=TUDelft/19_October_2008&action=edit 19] |
| [http://2008.igem.org/TUDelft/20_October_2008 20] | [http://2008.igem.org/TUDelft/21_October_2008 21] | [http://2008.igem.org/TUDelft/22_October_2008 22] | [http://2008.igem.org/TUDelft/23_October_2008 23] | [http://2008.igem.org/TUDelft/24_October_2008 24] | [http://2008.igem.org/wiki/index.php?title=TUDelft/25_October_2008&action=edit 25] | [http://2008.igem.org/wiki/index.php?title=TUDelft/26_October_2008&action=edit 26] |
| [http://2008.igem.org/wiki/index.php?title=TUDelft/27_October_2008&action=edit 27] | [http://2008.igem.org/wiki/index.php?title=TUDelft/28_October_2008&action=edit 28] | [http://2008.igem.org/wiki/index.php?title=TUDelft/29_October_2008&action=edit 29] | [http://2008.igem.org/wiki/index.php?title=TUDelft/30_October_2008&action=edit 30] | [http://2008.igem.org/wiki/index.php?title=TUDelft/31_October_2008&action=edit 31] | ||
Contents |
September 22nd 2008
3A Assembly
We've received our last thermopart from GeneArt and have started construction of the two remaining parts today. The parts under construction are now [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115030 K115030] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_K115035 K115035].
Growing induced cultures
We've started low temperature (26ºC) growing of induced strains today. Cultures grown contained either pSB1AT3 + P1010, K115012, K115029, K115031, K115032 or K115034. Some had different plasmids, either pSB1AT3 or pSB1AK3, so we've grown them on Ampicillin to reduce growing conditions variation. We aimed for lysis on Wednesday (OD=0.6). However that was planned for T=20ºC, and the temperature of our water bath could not go below 26ºC. We'll lyse the cells when they are ready.
Protein determination
To find better values to correct luciferase measurements for (instead of OD), we've measured total protein content. To make results easier to view we've made the following calculation: Correct luminescence for protein content by dividing each measured luminescence by the protein content of the sample. Normalize the luminescence of the thermosensitive construct K115034 for the luminescence of the control construct K115012. Results displayed in table 1 are the final outcomes for these normalized values per sample treatment. Please note that these first measurements are more important for optimalization of our protocols, not for reliable results of our constructs.
| Growth scheme | Lysis method | Protein content (ug/ml) | Normalized luciferase expression | |
|---|---|---|---|---|
| Induced: 20 h @ Tr | Sonification | 0.87 | 1.76 | |
| Induced: 20 h @ 37oC | Sonification | 1.88 | 0.35 | |
| Non induced: 15 h @ 37oC; induced: 5 h @ Tr | Sonification | 2.37 | 316.65 | |
| Induced: 40 hours @ Tr | Sonification | 2.47 | 0.09 | |
| Induced: 40 hours @ Tr | Promega Lysis buffer | 2.37 | 0.24 |
Much conclusions still can't be drawn on these data. There was an indication the R0080 arabinose induction might not be working properly. Either we'll have to use F2620 as promoter or just keep growth at the temperature desired. Another option might be to use the commercial pBAD vector.
