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Team:TUDelft/19 August 2009
From 2009.igem.org
(Difference between revisions)
| Line 87: | Line 87: | ||
| D2tr || TRI || 10<sup>-1</sup>|| overgrown | | D2tr || TRI || 10<sup>-1</sup>|| overgrown | ||
|- align="center" | |- align="center" | ||
| - | | D3tr || TRI || 10<sup>-2</sup>|| <font color=green><B>4450</b></font> | + | | [https://2009.igem.org/Image:Conjugation-aug18-trbK_D3tr.jpeg D3tr] || TRI || 10<sup>-2</sup>|| <font color=green><B>4450</b></font> |
|- align="center" | |- align="center" | ||
| D4tr || TRI || 10<sup>-3</sup>|| 714 | | D4tr || TRI || 10<sup>-3</sup>|| 714 | ||
| Line 93: | Line 93: | ||
| D5tr || TRI || 10<sup>-4</sup>|| 86 | | D5tr || TRI || 10<sup>-4</sup>|| 86 | ||
|- align="center" | |- align="center" | ||
| - | | T1tr || TRI + CAM || 10<sup>0</sup> || <font color=green><B>2</b></font> | + | | [https://2009.igem.org/Image:Conjugation-aug18-trbK_T1tr.jpeg T1tr] || TRI + CAM || 10<sup>0</sup> || <font color=green><B>2</b></font> |
|- align="center" | |- align="center" | ||
| T2tr || TRI + CAM || 10<sup>-1</sup>|| 0 | | T2tr || TRI + CAM || 10<sup>-1</sup>|| 0 | ||
| Line 119: | Line 119: | ||
| D7tr || TRI || 10<sup>-1</sup>|| overgrown | | D7tr || TRI || 10<sup>-1</sup>|| overgrown | ||
|- align="center" | |- align="center" | ||
| - | | D8tr || TRI || 10<sup>-2</sup>|| <font color=green><B>6930</b></font> | + | | [https://2009.igem.org/Image:Conjugation-aug18-trbK_D8tr.jpeg D8tr] || TRI || 10<sup>-2</sup>|| <font color=green><B>6930</b></font> |
|- align="center" | |- align="center" | ||
| D9tr || TRI || 10<sup>-3</sup>|| 720 | | D9tr || TRI || 10<sup>-3</sup>|| 720 | ||
| Line 125: | Line 125: | ||
| D10tr || TRI || 10<sup>-4</sup>|| 81 | | D10tr || TRI || 10<sup>-4</sup>|| 81 | ||
|- align="center" | |- align="center" | ||
| - | | T6tr || TRI + CAM || 10<sup>0</sup> || <font color=green><B>2</b></font> | + | | [https://2009.igem.org/Image:Conjugation-aug18-trbK_T6tr.jpeg T6tr] || TRI + CAM || 10<sup>0</sup> || <font color=green><B>2</b></font> |
|- align="center" | |- align="center" | ||
| T7tr || TRI + CAM || 10<sup>-1</sup>|| 0 | | T7tr || TRI + CAM || 10<sup>-1</sup>|| 0 | ||
Revision as of 15:36, 19 October 2009
Lab Notebook
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19 August 2009
Tim Weenink
After yesterdays horrible discovery that the I-SceI Homing Endonuclease we got from MIT was actually T4 DNA ligase we had to adjust our cloning strategy. The more primitive part without promoter was found to be the correct one, except that is was assembled with part !A instead of p(LacI). So we did that assembly again today. With p(LacI) from the delay team upstream, *T1 (downstream) and backbone pSB1C3 (also from the delay team). Both RbCl chemically comp and EC electrocomp cells were transformed.
Saeed
| lane | Part | Expected Plasmid Size | Status |
| 1 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ||
| 2 | !Fr1n upstream | 1900 | ok, but not needed |
| 3 | !Fr1n downstream | 1900 | ok |
| 4 | destination plasmid | 3000 | ok |
!Fr1n downstream will be purified from the gel by gel extraction and used for the new stategy as Tim mentioned before.
Daniel
Digestion of biobricks:
| Well | Biobrick | Expected Plasmid Size | Status |
| 1 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] | ||
| 2 | C0051 | 750 | ✔ |
| 3 | I13507 | 861 | ✔ |
| 4 | P0440 | 840 | ✔ |
| 5 | I13504 | 875 | ✔ |
| 6 | J04630 | 857 | ✔ |
| 7 | R0051 | 44 | too small |
| 8 | B0015 | 129 | ✔ |
| 9 | J13002 | 74 | ✔ |
| 10 | pSB1C3 | 2072 | ✔ |
| 11 | pSB1AK3 | 3426 | ✔ |
| 12 | pSB1A3 | 2157 | ✔ |
| 13 | CB | ||
| 14 | CC | ||
| 15 | [http://www.eurogentec.com/EGT/Images/RESALES/Electrophoresis/Regular%20DNA%20Ladder/7-SmartLadder.jpg DNA Ladder] |
Assemblies 1A, 2A, 3A, 4A, 5A and 6A
Transformation and culture in plates and tubes
Calin
Good results on the conjugation test. All plates were imaged on the safe imager with the 5 Megapixel camera. Colony counting was done with [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1770926 Clono-Counter].
For the oriTR conjugation test:
| Set 1 | |||
| Plate ID | Antibiotics | Dilution | # Colonies |
| D1tr | TRI | 100 | overgrown |
| D2tr | TRI | 10-1 | overgrown |
| D3tr | TRI | 10-2 | 4450 |
| D4tr | TRI | 10-3 | 714 |
| D5tr | TRI | 10-4 | 86 |
| T1tr | TRI + CAM | 100 | 2 |
| T2tr | TRI + CAM | 10-1 | 0 |
| T3tr | TRI + CAM | 10-2 | 0 |
| T4tr | TRI + CAM | 10-3 | 0 |
| T5tr | TRI + CAM | 10-4 | 0 |
| R1tr | CAM | 100 | overgrown |
| R2tr | CAM | 10-1 | 8636 |
| Set 2 | |||
| Plate ID | Antibiotics | Dilution | # Colonies |
| D6tr | TRI | 100 | overgrown |
| D7tr | TRI | 10-1 | overgrown |
| D8tr | TRI | 10-2 | 6930 |
| D9tr | TRI | 10-3 | 720 |
| D10tr | TRI | 10-4 | 81 |
| T6tr | TRI + CAM | 100 | 2 |
| T7tr | TRI + CAM | 10-1 | 0 |
| T8tr | TRI + CAM | 10-2 | 0 |
| T9tr | TRI + CAM | 10-3 | 0 |
| T10tr | TRI + CAM | 10-4 | 0 |
| R3tr | CAM | 100 | overgrown |
| R4tr | CAM | 10-1 | 6333 |
also 3 positive control plates with no TrbK expression.
For the oriTR conjugation test:
| Set 1 | |||
| Plate ID | Antibiotics | Dilution | # Colonies |
| D1ori | TRI + CAM | 100 | overgrown |
| D2ori | TRI + CAM | 10-1 | overgrown |
| D3ori | TRI + CAM | 10-2 | 2939 |
| D4ori | TRI + CAM | 10-3 | 470 |
| D5ori | TRI + CAM | 10-4 | 48 |
| Ts1ori | AMP + CAM | 100 | 520 |
| Ts2ori | AMP + CAM | 10-1 | 1 |
| Ts3ori | AMP + CAM | 10-2 | 0 |
| Ts4ori | AMP + CAM | 10-3 | 0 |
| Ts5ori | AMP + CAM | 10-4 | 0 |
| TL1ori | TRI + AMP | 100 | 4092 |
| TL2ori | TRI + AMP | 10-1 | 802 |
| TL3ori | TRI + AMP | 10-2 | 94 |
| TL4ori | TRI + AMP | 10-3 | 7 |
| TL5ori | TRI + AMP | 10-4 | 0 |
| R1ori | AMP | 100 | overgrown |
| R2ori | AMP | 10-1 | 5187 |
| Set 2 | |||
| Plate ID | Antibiotics | Dilution | # Colonies |
| D6ori | TRI + CAM | 100 | overgrown |
| D7ori | TRI + CAM | 10-1 | overgrown |
| D8ori | TRI + CAM | 10-2 | 4950 |
| D9ori | TRI + CAM | 10-3 | 1066 |
| D10ori | TRI + CAM | 10-4 | 114 |
| Ts6ori | AMP + CAM | 100 | 1105 |
| Ts7ori | AMP + CAM | 10-1 | 12 |
| Ts8ori | AMP + CAM | 10-2 | 0 |
| Ts9ori | AMP + CAM | 10-3 | 0 |
| Ts10ori | AMP + CAM | 10-4 | 0 |
| TL6ori | TRI + AMP | 100 | overgrown |
| TL7ori | TRI + AMP | 10-1 | 2251 |
| TL8ori | TRI + AMP | 10-2 | 352 |
| TL9ori | TRI + AMP | 10-3 | 39 |
| TL10ori | TRI + AMP | 10-4 | 7 |
| R3ori | AMP | 100 | overgrown |
| R4ori | AMP | 10-1 | 3961 |
| Set 3 | |||
| Plate ID | Antibiotics | Dilution | # Colonies |
| D11ori | TRI + CAM | 100 | overgrown |
| D12ori | TRI + CAM | 10-1 | overgrown |
| D13ori | TRI + CAM | 10-2 | 4180 |
| D14ori | TRI + CAM | 10-3 | 737 |
| D15ori | TRI + CAM | 10-4 | 46 |
| Ts11ori | AMP + CAM | 100 | bad plate |
| Ts12ori | AMP + CAM | 10-1 | 3 |
| Ts13ori | AMP + CAM | 10-2 | 0 |
| Ts14ori | AMP + CAM | 10-3 | 0 |
| Ts15ori | AMP + CAM | 10-4 | 0 |
| TL11ori | TRI + AMP | 100 | overgrown |
| TL12ori | TRI + AMP | 10-1 | 1436 |
| TL13ori | TRI + AMP | 10-2 | 172 |
| TL14ori | TRI + AMP | 10-3 | 18 |
| TL15ori | TRI + AMP | 10-4 | 3 |
| R5ori | AMP | 100 | overgrown |
| R6ori | AMP | 10-1 | 3679 |
