June 19
Collection of soil and water samples from LA River
June 20
Lab walkthrough
Setting up lab, stock supplies, preparation of LB-amp plates
Test extraction of DNA from BioBrick wells into cloning plates ([http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_K123001 K123001], [http://partsregistry.org/Part:BBa_K123002 K123002], [http://partsregistry.org/Part:BBa_K123003 K123003])
Initial enrichment cultures of collected samples on minimal media with BPA or 17a-ethynylestradiol
June 21
MoBio Powermax Soil kit extraction of DNA from collected samples
Initial enrichment cultures of samples in minimal media with nonylphenol or DDT
Geneious and primer design tutorial
Results
MoBio Extraction
| Sample |
Concentration (ng/µl) |
| 1 |
4.5 |
| 2 |
4.1 |
| 4 |
0.0 |
| 5 |
0.7 |
| 6 |
13.7 |
| 7 |
5.8 |
| 9 |
58.7 |
| 10 |
11.4 |
Locations 3 and 8 were omitted from the MoBio Powermax Soil Extraction procedure because they were liquid samples.
June 22
Miniprep and sequencing of selected BioBricks (BBa_K123000, BBa_K123001, BBa_K123002, BBa_K123003)
Preparation of competent cells
Results of OD measurements:
| Part |
Concentration (ng/µl) |
| K123000 |
326.6 |
| K123001 |
293.8 |
| K123002 |
152.3 |
| K123003 |
402.6 |
June 23
Transformation of T7 polymerase ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]]), and double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) biobricks for test sequences for BisA and BisB degradation genes ([http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001])
Retested competent cells made June 22nd with [http://partsregistry.org/Part:BBa_K123001 K123001] and pUC
Transfered 0.5 mL aliquots of BPA and 5mL 17a-ethynylestradiol cultures from June 20 to new tubes of minimal media
Designed primers for test sequence of BisdA (pNT001)
Designed primers to continue sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003])
We determined that sequences for [http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001] have correct protein coding but different codons than in the sequences shown online
June 24
Transformation of Tet repressible promoter [http://partsregistry.org/Part:BBa_R0040 R0040]
Preparation of LB, LB (no antibiotic) plates, and SOC media
Transfer of 5 mL DDT and nonylphenol cultures from June 21 to new tubes of minimal media
Design of reverse primer for continued sequencing of human ER ([http://partsregistry.org/Part:BBa_K123003 K123003])
Update Wiki with protocols we have been using
We determined that our competent cells are functional have a cfu/ug DNA of 6.6 x 10^6.
June 25
Take out plates from June 24
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