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
Initial enrichment cultures of collected samples on minimal media with BPA or 17a-ethynylestradiol
June 21
MoBio kit extraction of DNA from collected samples
Initial enrichment cultures of samples in minimal media with nonylphenol or DDT
Geneious and primer design tutorial
June 22
Miniprep and sequencing of selected BioBricks (K123000, K123001, K123002, K123003)
Preparation of competent cells
Results
| Part |
Concentration |
| K123000 |
326.6 |
| K123001 |
293.8 |
| K123002 |
152.3 |
| K123003 |
402.6 |
June 23
Transform T7 polymerase (K145001), mCherry (J06702]), Lac Promoter (R0010]), and double terminator (B0014 and B0015) biobricks for test sequences for BisA and BisB degradation genes (K123000 and K123001)
Retest competent cells made June 22nd with K123001 and pUC
Transfer 0.5 mL aliquots of BPA and 5mL 17a-ethynylestradiol cultures from June 20 to new tubes of minimal media
Design primers for test sequence of BisdA (pNT001)
Design primers to continue sequencing of human ER (K123003)
Results
We determined that sequences for K123000 and K123001 have correct protein coding but different codons than in the sequences shown online
June 24
Transform Tet repressible promoter R0040
Make plain LB and pour plain LB plates
Make SOC media
Transfer 5 mL DDT and nonylphenol cultures from June 21 to new tubes of minimal media
Wash dishes from last year
Design reverse primer for continued sequencing of human ER (K123003)
Update Wiki with protocols we have been using
Results
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
"
