Team:Caltech/Week 10

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Caltech iGEM 2011

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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

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.

August 20

Visit West Basin Municipal Water Facility