"

From 2011.igem.org

Revision as of 16:05, 5 October 2011 (view source) Silu (Talk | contribs) ← Older edit		Revision as of 01:35, 6 October 2011 (view source) CubicStone (Talk | contribs) Newer edit →
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

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.