Team:TUDelft/6 August 2009
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='''6 August 2009'''= | ='''6 August 2009'''= | ||
+ | ===Sriram=== | ||
+ | Today I continued with the ligation of the restricted parts done yesterday. Then the ligated parts were electro transformed and also heat shock transformed. There are in total 8 assembly as follows: <br> | ||
+ | 1. pTet + RBS-cI-RBS (Chloramphenicol backbone) <br> | ||
+ | 2. mRFP + Double Terminator (Chloramphenicol backbone) <br> | ||
+ | 3. pLacI + RBS (Chloramphenicol backbone) <br> | ||
+ | 4. TetR + Double Terminator (Chloramphenicol backbone) <br> | ||
+ | 5. pTet + Lock3c (Chloramphenicol backbone) <br> | ||
+ | 6. cI + Double Terminator (Chloramphenicol backbone) <br> | ||
+ | 7. λp-in + RBS-GFP-Double Term (Chloramphenicol backbone) <br> | ||
+ | 8. pLacI + key3c (Amp-Kan backbone) <br> | ||
===Calin=== | ===Calin=== | ||
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| D1 || TRI || 10<sup>0</sup> || overgrown | | D1 || TRI || 10<sup>0</sup> || overgrown | ||
|- align="center" | |- align="center" | ||
- | | D2 || TRI || 10<sup>-2</sup>|| <font color=green><B>6770</b></font> | + | | [https://2009.igem.org/Image:TUDelft-aug5-2009-conj-test-plate-D2-noGFP.jpg D2] || TRI || 10<sup>-2</sup>|| <font color=green><B>6770</b></font> |
|- align="center" | |- align="center" | ||
| D3 || TRI || 10<sup>-4</sup>|| 170 | | D3 || TRI || 10<sup>-4</sup>|| 170 | ||
Line 31: | Line 41: | ||
| T2 || TRI + AMP || 10<sup>-1</sup>|| ? | | T2 || TRI + AMP || 10<sup>-1</sup>|| ? | ||
|- align="center" | |- align="center" | ||
- | | T3 || TRI + AMP || 10<sup>-2</sup>|| <font color=green><B>186</b></font> | + | | [https://2009.igem.org/Image:TUDelft-aug5-2009-conj-test-plate-T3-GFP.jpg T3] || TRI + AMP || 10<sup>-2</sup>|| <font color=green><B>186</b></font> |
|- align="center" | |- align="center" | ||
| T4 || TRI + AMP || 10<sup>-3</sup>|| 11 | | T4 || TRI + AMP || 10<sup>-3</sup>|| 11 | ||
Line 59: | Line 69: | ||
| D6 || TRI || 10<sup>0</sup> || overgrown | | D6 || TRI || 10<sup>0</sup> || overgrown | ||
|- align="center" | |- align="center" | ||
- | | D7 || TRI || 10<sup>-2</sup>|| <font color=green><B>8320</b></font> | + | | [https://2009.igem.org/Image:TUDelft-aug5-2009-conj-test-plate-D7-noGFP.jpg D7] || TRI || 10<sup>-2</sup>|| <font color=green><B>8320</b></font> |
|- align="center" | |- align="center" | ||
| D8 || TRI || 10<sup>-4</sup>|| 114 | | D8 || TRI || 10<sup>-4</sup>|| 114 | ||
Line 71: | Line 81: | ||
| T9 || TRI + AMP || 10<sup>-1</sup>|| ? | | T9 || TRI + AMP || 10<sup>-1</sup>|| ? | ||
|- align="center" | |- align="center" | ||
- | | T10 || TRI + AMP || 10<sup>-2</sup>|| <font color=green><B>228</b></font> | + | | [https://2009.igem.org/Image:TUDelft-aug5-2009-conj-test-plate-T10-GFP.jpg T10] || TRI + AMP || 10<sup>-2</sup>|| <font color=green><B>228</b></font> |
|- align="center" | |- align="center" | ||
| T11 || TRI + AMP || 10<sup>-3</sup>|| 18 | | T11 || TRI + AMP || 10<sup>-3</sup>|| 18 | ||
Line 91: | Line 101: | ||
| D11 || TRI || 10<sup>0</sup> || overgrown | | D11 || TRI || 10<sup>0</sup> || overgrown | ||
|- align="center" | |- align="center" | ||
- | | D12 || TRI || 10<sup>-2</sup>|| <font color=green><B>8950</b></font> | + | | [https://2009.igem.org/Image:TUDelft-aug5-2009-conj-test-plate-D12-noGFP.jpg D12] || TRI || 10<sup>-2</sup>|| <font color=green><B>8950</b></font> |
|- align="center" | |- align="center" | ||
| D13 || TRI || 10<sup>-4</sup>|| 173 | | D13 || TRI || 10<sup>-4</sup>|| 173 | ||
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| T16 || TRI + AMP || 10<sup>-1</sup>|| ? | | T16 || TRI + AMP || 10<sup>-1</sup>|| ? | ||
|- align="center" | |- align="center" | ||
- | | T17 || TRI + AMP || 10<sup>-2</sup>|| <font color=green><B>218</b></font> | + | | [https://2009.igem.org/Image:TUDelft-aug5-2009-conj-test-plate-T17-GFP.jpg T17] || TRI + AMP || 10<sup>-2</sup>|| <font color=green><B>218</b></font> |
|- align="center" | |- align="center" | ||
| T18 || TRI + AMP || 10<sup>-3</sup>|| 31 | | T18 || TRI + AMP || 10<sup>-3</sup>|| 31 | ||
Line 133: | Line 143: | ||
Elecroporated 10 assemblies. Experienced some arcing with assemblies 1 and 6. Assembly 6 was redone with only 2 uL of DNA added. | Elecroporated 10 assemblies. Experienced some arcing with assemblies 1 and 6. Assembly 6 was redone with only 2 uL of DNA added. | ||
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===Tim Weenink=== | ===Tim Weenink=== |
Latest revision as of 08:01, 20 October 2009
Lab Notebook
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6 August 2009
Sriram
Today I continued with the ligation of the restricted parts done yesterday. Then the ligated parts were electro transformed and also heat shock transformed. There are in total 8 assembly as follows:
1. pTet + RBS-cI-RBS (Chloramphenicol backbone)
2. mRFP + Double Terminator (Chloramphenicol backbone)
3. pLacI + RBS (Chloramphenicol backbone)
4. TetR + Double Terminator (Chloramphenicol backbone)
5. pTet + Lock3c (Chloramphenicol backbone)
6. cI + Double Terminator (Chloramphenicol backbone)
7. λp-in + RBS-GFP-Double Term (Chloramphenicol backbone)
8. pLacI + key3c (Amp-Kan backbone)
Calin
Good results on the conjugation test. Many colonies. All plates were imaged on the safe imager with the 5 Megapixel camera. The plates with GFP (T and R) were excited by blue light and placed under a filter (50 ms exposure). The plates without GFP (D) were imaged with the upper white light and an exposure of 15ms. Images were color inverted before the image analysis. Colony counting was done with [http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1770926 Clono-Counter].
Set 1 | |||
Plate ID | Antibiotics | Dilution | # Colonies |
D1 | TRI | 100 | overgrown |
D2 | TRI | 10-2 | 6770 |
D3 | TRI | 10-4 | 170 |
D4 | TRI | 10-5 | 40 |
D5 | TRI | 10-6 | 10 |
T1 | TRI + AMP | 100 | ? |
T2 | TRI + AMP | 10-1 | ? |
T3 | TRI + AMP | 10-2 | 186 |
T4 | TRI + AMP | 10-3 | 11 |
T5 | TRI + AMP | 10-4 | 0 |
T6 | TRI + AMP | 10-5 | 0 |
T7 | TRI + AMP | 10-6 | 0 |
R1 | AMP | 100 | overgrown |
R2 | AMP | 10-4 | 32 |
R3 | AMP | 10-5 | 6 |
R4 | AMP | 10-6 | 2 |
Set 2 | |||
Plate ID | Antibiotics | Dilution | |
D6 | TRI | 100 | overgrown |
D7 | TRI | 10-2 | 8320 |
D8 | TRI | 10-4 | 114 |
D9 | TRI | 10-5 | 18 |
D10 | TRI | 10-6 | 8 |
T8 | TRI + AMP | 100 | ? |
T9 | TRI + AMP | 10-1 | ? |
T10 | TRI + AMP | 10-2 | 228 |
T11 | TRI + AMP | 10-3 | 18 |
T12 | TRI + AMP | 10-4 | 2 |
T13 | TRI + AMP | 10-5 | 0 |
T14 | TRI + AMP | 10-6 | 0 |
Set 3 | |||
Plate ID | Antibiotics | Dilution | |
D11 | TRI | 100 | overgrown |
D12 | TRI | 10-2 | 8950 |
D13 | TRI | 10-4 | 173 |
D14 | TRI | 10-5 | 22 |
D15 | TRI | 10-6 | 1 |
T15 | TRI + AMP | 100 | ? |
T16 | TRI + AMP | 10-1 | ? |
T17 | TRI + AMP | 10-2 | 218 |
T18 | TRI + AMP | 10-3 | 31 |
T19 | TRI + AMP | 10-4 | 2 |
T20 | TRI + AMP | 10-5 | 0 |
T21 | TRI + AMP | 10-6 | 0 |
Calculated conjugation efficiencies:
Set | Conjugation Efficiency |
1 | 0.028 |
2 | 0.028 |
3 | 0.025 |
Trimethoprim control plate had no growth.
5mL tube of assembly CA made.
Elecroporated 10 assemblies. Experienced some arcing with assemblies 1 and 6. Assembly 6 was redone with only 2 uL of DNA added.
Tim Weenink
Elecroporated 10 assemblies.
Biobrick | origin of parts |
Orr
Made 2 one liter batches of LB medium, and prepared some chemically competent TSS cells.