July 3
Checked Gibson transformations
Plated remaining transformed cells
Made new dilutions of original enrichment cultures (BPA, nonylphenol, 17-alpha-ethinyl-estradiol, DDT)
Results
There was no growth on the transformed Gibson plates. Since we did a small reaction, we used all of the reaction in transforming competent cells. The Gibson assembly likely failed, as one of the negative control plates had a single colony. However, we have had trouble with our competent cells in the past and will try plating what we have left.
Gibson Transformations
| Plate |
Number of Colonies |
| pNT001 + |
0 |
| pNT001 - |
0 |
| pNT002 + |
0 |
| pNT002 - |
1 |
July 4
Results
After plating the remaining 450 ul of our transformed cells:
| Plate |
Number of Colonies |
| pNT001 + |
10 |
| pNT001 - |
0 |
| pNT002 + |
1 |
| pNT002 - |
0 |
This Week
Start enrichment cultures from a previous iteration, as the latest dilutions remain clear.
Design experiments to test for degradation of BPA by pNT001 and pNT002 using the HPLC
Get the BioBricks we ordered from the parts registry ([http://partsregistry.org/Part:BBA_K364309 K364309], [http://partsregistry.org/Part:BBA_K364329 K364329], [http://partsregistry.org/Part:BBA_K364327 K364327], [http://partsregistry.org/Part:BBA_K364307 K364307])
Transform these into cells, miniprep and sequence
Continue with enrichment cultures
Put LA River soil DNA into fosmids
Work on Project Description and Safety Proposal
Once we've sequenced ER ([http://partsregitry.org/Part:BBa_K123003 K123003]), start making test plasmids to see if it works.
July 5
Start overnight cultures of Gibson transformations
Start gel to figure out length of purified river DNA (for fosmid kit)
Confirm we have enough DNA for work with estrogen receptor
Streak out new biobricks on chloramphenicol plates
Results
The gel of the river DNA was inconclusive, as no bands were visible (even the weight ladder). The gel will be rerun.
July 6
Miniprep Gibson transformations and send off for sequencing
Redo gel for fosmid kit
Continue sequencing of estrogen receptor (using new forward primer and old reverse primer)
Start overnight cultures of new biobricks
Plate enrichment cultures on LB
Results
Again, no bands were visible on the gel. We will run the samples on a smaller gel to check the size of the DNA and confirm that there is in fact DNA in the sample.
July 7
Miniprep new biobricks and send off for sequencing
Meet with Pedro about HPLC and p450s
Image enrichment culture plates
Image second fosmid kit gel
Run river DNA on small gel to check size
Start new minimal media cultures from plates
Results
The smaller gel shows a smear at the top of the gel for one of the samples, indicating that this sample contains DNA that is probably of the correct size for work with the fosmid kit.
File:River gel small.tif
All of the plates of the enrichment cultures showed growth.
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