Team:Tsinghua-A/Wetlab

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Revision as of 02:48, 28 October 2011

JavaScript Slideshow - TinySlideshow



Contents

Parts List


Name

Type

Description

Length

BBa_K574000

Composite

pLux + TetR

903

BBa_K574001

Composite

TetR regulated by 3OC6HSL

1890

BBa_K574002

Composite

pluxR + CFP

941

BBa_K574003

Device

3OC6HSL -> TetR && reporter

2839

BBa_K574004

Composite

3OC12HSL regulated by TetR

797

BBa_K574005

Composite

3OC12HSL and YFP regulated by pBad

1712

BBa_K574006

Device

3OC12HSL regulated by pTet and pBad

2517

BBa_K574007

Composite

lasR + TT

918

BBa_K574008

Composite

a constitutive LasR producer

982

BBa_K574009

Composite

3OC12HSL -> PoPS Receiver

1147

Descriptions


We assembled BBa_E0840 under the pLas promoter BBa_R0079 that was contained into K574009. We kept pSB1A2 as the scaffold vector.


Methods


1. Ligate BBa_E0840 downstream of BBa_K574009 , which acts as a reporter.

2. Culture the positive colony at 37°C, 220 rpm for 12 hours, as well as the DH5alpha.

3. Dilute 1:25 the former in 15 falcon tubes (4 ml cultures), each 3 tubes as a group. The later is also diluted in 3 tubes as the negative control.

4. After culturing for another 2 hours, as their OD600 reach 0.2, induce with 3OC12HSL 0, 1e-7 M, 1e-6 M, 1e-5 M and 1e-4 M respectively.

5. Take samples after 1 hour, 4 hours, 8 hours and 24 hours, that is, centrifugalizate 0.5 mL of each culture and suspend them by 1 mL of PBS (phosphate-buffered saline).

6. Measure the fluorescence of the samples with the flow cytometer.


Results


From the chart, we can see that cells were hardly induced in the control group, and with the concentration of inducer growing, the intensity of GFP increased by groups. The most efficient concentration of inducer was around 10-5M, and higher concentration may lead to the expression of GFP decreasing. Additionally, to most groups, the intensity of GFP reached its maxium after 4 hours.




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