Team:Caltech/Week 4

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

July 4

Results

After plating the remaining 450 ul of our transformed cells:

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
Design primers for estrogen receptor test construct

Results

River samples run on a small gel. 1: ladder; 2:blank; 3: sample 9; 4: sample 10

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.

All of the plates of the enrichment cultures showed growth.

LB Plates of Enrichment Cultures

DDT with vitamins	DDT without vitamins	17a-ethynylestradiol with vitamins	17a-ethynylestradiol without vitamins
Nonylphenol with vitamins	Nonylphenol without vitamins	BPA with vitamins, room temperature, locations 1 and 2	BPA with vitamins, room temperature, locations 3 and 4
BPA without vitamins, room temperature, locations 1 and 2	BPA without vitamins, room temperature, locations 3 and 4	BPA with vitamins, 30˚C, locations 1 and 2	BPA with vitamins, 30˚C, locations 3 and 4
BPA without vitamins, 30˚C, locations 1 and 2	BPA without vitamins, 30˚C, locations 3 and 4

July 8

Send off pNT001 and pNT002 for sequencing again
Talk to Kenny about PFGE
Work on design of test constructs for estrogen receptor

July 10

Start overnight culture of pSB4A5 for use in construction of pNT003