Team:CSU Fort Collins/Notebook

March:

3/12/15:

Made overnight cultures of lac promoter in pSB1C3 and of KillerRed and trans-zeatin in backbones from synthesis.

3/13/15:

Prepared minipreps from overnight cultures of lac promoter, KillerRed, and trans-zeatin plasmids.

3/14/15:

Completed digestion, ligation, and transformation of E. coli with lac:KillerRed and lac:trans-zeatin.

3/17/15:

Checked colonies with colony PCR protocol. Used annealing temperature of 65℃ and extension time of 2 minutes. Ran gel of results. lac:KillerRed colonies 4, 9 were successful.

From left to right: Ladder 1, KillerRed colonies 1-9, ladder 2, trans-zeatin colonies 10-16, positive control

3/18/15:

Miniprepped lac:KillerRed colonies from overnight cultures using kit instructions. Nanodropped results. Re-tried construction of lac:trans-zeatin.

3/19/15:

Checked colonies of lac:trans-zeatin. Results successful.

May:

5/15/15:

Miniprepped overnight cultures of KillerRed in pSB1C3. Attempted to create RBS:KillerRed using SLIM procedure. Made new LB, LB + Amp100 plates, and LB + Kn50 plates.

5/18/15:

Checked May 15th results for RBS:KillerRed on gel; results unsuccessful. Retried procedure using a different thermocycler.

June:

6/15/15:

Prepare media and overnight cultures for trans-zeatin growth experiment.

6/16/15:

Followed procedure for trans-zeatin growth experiment up to a final growth time of 1 day.

6/18/15:

Ran HPLC procedure for analysis of fermentative products.

July:

7/17/15:

Created media and prepared flasks for trans-zeatin growth experiment. The experiment needed to be repeated because we suspected that 1 day was not long enough for the trans-zeatin to be created in detectable amounts.

7/19/15:

Created overnight cultures of strains for trans-zeatin growth experiment.

7/20/15:

Day 1 of trans-zeatin growth experiment.

7/21/15:

Day 2 of trans-zeatin growth experiment.

7/22/15:

Day 3 of trans-zeatin growth experiment. Collected final samples of liquid culture. Cells were accidentally thrown away, so the experiment needed to be repeated.

August:

8/19/15:

Made media, autoclaved flasks, and prepared overnight cultures to run trans-zeatin growth experiment.

8/20/15:

Day 1 of trans-zeatin growth experiment.

8/21/15:

Day 2 of trans-zeatin growth experiment.

8/22/15:

Day 3 of trans-zeatin growth experiment.

September:

9/7/15:

Made media and prepared bioreactors for trans-zeatin prototype (bioreactor experiment).

9/10/15:

Made 50 mL overnight cultures for bioreactor experiment.

9/11/15:

Day 1 of bioreactor experiment.

9/12/15:

Day 2 of bioreactor experiment.

9/13/15:

Day 3 of bioreactor experiment. Took samples and extracted zeatin from bioreactor and previous experimental results using single-phase extraction procedure.

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