Team:Tsinghua-A/Parts

From 2011.igem.org

(Difference between revisions)
Line 288: Line 288:
which acts as a reporter.<BR>2. Culture the positive colony at 37&deg;C,
which acts as a reporter.<BR>2. Culture the positive colony at 37&deg;C,
220 rpm for 12 hours, as well as the DH5alpha.<BR>3. Dilute 1:25 the
220 rpm for 12 hours, as well as the DH5alpha.<BR>3. Dilute 1:25 the
-
former in 12 falcon tubes (4 ml cultures), each 3 tubes as a group.
+
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.<BR>4.
The later is also diluted in 3 tubes as the negative control.<BR>4.
After culturing for another 2 hours, as their OD600 reach 0.2, induce
After culturing for another 2 hours, as their OD600 reach 0.2, induce
-
with 3OC12HSL 0, 1e-6 M, 1e-5 M and 1e-4 M respectively.<BR>5. Take
+
with 3OC12HSL 0, 1e-7 M, 1e-6 M, 1e-5 M and 1e-4 M respectively.<BR>5. Take
samples after 1 hour, 4 hours, 8 hours and 24 hours, that is,
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
centrifugalizate 0.5 mL of each culture and suspend them by 1 mL of
Line 309: Line 309:
group, and with the concentration of inducer growing, the intensity
group, and with the concentration of inducer growing, the intensity
of GFP increased by groups. The most efficient concentration of
of GFP increased by groups. The most efficient concentration of
-
inducer was around 10^-6M, and to most groups, the intensity of GFP
+
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.</SPAN></FONT></FONT></FONT></P>
reached its maxium after 4 hours.</SPAN></FONT></FONT></FONT></P>

Revision as of 01:35, 6 October 2011

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Parts Section



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



Description:

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.