August 15
CPCR of Gibson pNT002 and pNT003
Miniprep J23119 and I732019 to construct LacZ producer
GCMS of bisphenol A dissolved in methanol
Gibson 16s sequencing DNA and vector together
Results
CPCR gels showed 200 bp bands, indicating self-ligation of all colonies picked.
GCMS results show no correct mass spectrometry peaks.
August 16
Electrospray bisphenol A dissolved in methanol
Attempt to standard assemble constitutively expressed LacZ and transform into XL10 gold. However, there was no good way to select out the lacZ insert (I732019) from the backbone it was cut out of, as they are the same length.
Nanodrop 16s solutions
Results
Electrospray results show correct mass spectrometry peaks
| Name |
concentration (ng/ul) |
| 9-3A |
35 |
| 9-3B |
47.9 |
| 9-9A |
79.5 |
| 9-9B |
50.4 |
| 9-9C |
40.4 |
| 10-3A |
31.1 |
August 17
Borrowed HindIII from Nate to be able to gel extract lacZ from the now smaller backbone pieces
Transformed psB1C3 into top10 cells
Results
The lacZ plated on LB-amp X-gal had about 520 colonies. The negative control had around 700 colonies. None of the colonies were blue, likely indicating that there was no functioning lacZ. Some were sent off for sequencing.
Colonies for 16s sequencing!
- 9-3A (1)
- 9-3B (12)
- 9-9A (1)
- 9-9B (1)
- 9-9C (0)
- 10-3A (1)
August 18
Restriction digest of R0010 and J23119 and their backbone for lacZ construct. Ligation of each promoter/backbone with lacZ purified yesterday. Transformed in XL-10 gold
Prepare p450-degraded BPA for electrospray using organic extraction
Start overnight cultures of 16s seequencing --> miniprep --> send off for sequencing
Begin overnight cultures of WT-F87A
Results
4 colonies on pSB1C3 plate, all pink.
August 19
Visit Monk Hill Groundwater Treatment Plant
Plate and sequence 16s vectors and WT-F87A
Make glycerol stocks of WT-F87A
Made new DH5a electrocompetent cells with Nate
Results
Electrospray of BPA in MeOH and its degraded products show a mass reduction of 22
Nanodrop:
| Name |
concentration (ng/ul) |
| 9-3A 1 |
32 |
| 9-3B 1 |
84.5 |
| 9-3B 2 |
46.7 |
| 9-3B 3 |
26.5 |
| 9-3B 4 |
57.5 |
| 9-3B 5 |
56.3 |
| 9-3B 6 |
70.1 |
| 9-3B 7 |
45 |
| 9-3B 8 |
59.9 |
| 9-3B 9 |
37.3 |
| 9-3B 10 |
29 |
| 9-3B 11 |
18.3 |
| 9-3B 12 |
52.6 |
| 9-9A 1 |
68.7 |
| 10-3A 1 |
17.6 |
| WT-F87A |
107.6 |
Number of colonies on X-gal-ampicillin-LB plates
| Name |
Number of colonies |
| J23119+lacZ + |
~1100 |
| J23119+lacZ - |
25 |
| R0010+lacZ + |
~1800 |
| R0010+lacZ- |
57 |
Most of the colonies on the positive plates were blue, indicating the presence of working lacZ.
LacZ with inducible promoter in XL-10 gold cells
|
LacZ with constitutive promoter in XL-10 gold cells
|
August 20
Visit West Basin Municipal Water Facility
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