Team:Caltech/Week 2

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Caltech iGEM 2011

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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 ([http://partsregistry.org/Part:BBa_K123000 K123000], [http://partsregistry.org/Part:BBa_K123001 K123001], [http://partsregistry.org/Part:BBa_K123002 K123002], [http://partsregistry.org/Part:BBa_K123003 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 ([http://partsregistry.org/Part:BBa_K145001 K145001]), mCherry ([http://partsregistry.org/Part:BBa_J06702 J06702]]), Lac Promoter ([http://partsregistry.org/Part:BBa_R0010 R0010]]), and double terminator ([http://partsregistry.org/Part:BBa_B0014 B0014] and [http://partsregistry.org/Part:BBa_B0015 B0015]) biobricks for test sequences for BisA and BisB degradation genes ([http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001])
Retest competent cells made June 22nd with [http://partsregistry.org/Part:BBa_K123001 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 ([http://partsregistry.org/Part:BBa_K123003 K123003])

Results

We determined that sequences for [http://partsregistry.org/Part:BBa_K123000 K123000] and [http://partsregistry.org/Part:BBa_K123001 K123001] have correct protein coding but different codons than in the sequences shown online

June 24

Transform Tet repressible promoter [http://partsregistry.org/Part:BBa_R0040 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 ([http://partsregistry.org/Part:BBa_K123003 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