Part:BBa_K176144

aTc&AHL->GFP: pCon 0.01->tetR-LVA+pCon 0.36->luxR+pLux/Tet->GFP

Input aTc activates GFP. Input AHL activates GFP.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 7
Illegal NheI site found at 30
Illegal NheI site found at 898
Illegal NheI site found at 921
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1876
Illegal BsaI.rc site found at 2620

MEARSUREMENT

We test the response of the hybrid promoter( K176000) to both tetR and AHL. Here LuxR is ligated after J23107.

INPUT : constitutive promoters +tetR , AHL
DEVICE: K176136( J23101 + K176126);
K176140( J23115 + K176126);
K176144( J23103 + K176126);
OUTPUT: GFP

Model

To see the details of the model, click [http://2009.igem.org/Team:USTC/Modeling/Model-3 here] please.

data

K176144

AHL(M)	T(h)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)
0	0.5	0.210333333	4.745	22.55942948	90.16558544
1.00E-10	0.5	0.245	7.730333333	31.55238095	126.1086369
1.00E-09	0.5	0.246333333	11.12366667	45.15696888	180.4834887
1.00E-08	0.5	0.25	11.69366667	46.77466667	186.9491074
1.00E-07	0.5	0.256666667	13.35266667	52.02337662	207.9271648
1.00E-05	0.5	0.249	14.77833333	59.35073628	237.2131746
0	1.5	0.430666667	10.12166667	23.50232198	93.93414061
1.00E-10	1.5	0.449	16.77366667	37.35783222	149.3118794
1.00E-09	1.5	0.441333333	133.0933333	301.570997	1205.319732
1.00E-08	1.5	0.429666667	189	439.8758728	1758.097013
1.00E-07	1.5	0.408333333	168.8066667	413.4040816	1652.294491
1.00E-05	1.5	0.38	138.9433333	365.6403509	1461.39229
0	3	0.666	20.16933333	30.28428428	121.0403049
1.00E-10	3	0.676	37.01733333	54.75936884	218.8623854
1.00E-09	3	0.648666667	451.2633333	695.6783145	2780.488867
1.00E-08	3	0.638333333	774.2666667	1212.950392	4847.923228
1.00E-07	3	0.561666667	580.5466667	1033.614243	4131.152052
1.00E-05	3	0.464333333	360.73	776.8772434	3105.024953

For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

Figure 3- this steregram shows the response of K176144 to both time and AHL. From this gram, we can see that the GFP expression reaches its top at some definite AHL(1.E-08) concentration at any time in the range of allowable error.

Figure 4- the steregram shows the response of different device( K176136; K176140; K176144 ) to both tetR and AHL. In this gram, the score( click here to see the details) represents the relative strength of different promoter before tetR-LVA, that is , to represent the INPUT of tetR.

protocol

AHLHybrid promoter:BBa_K176026, BBa_K176126, BBa_K176128, BBa_K176130

1. Streak a plate of the strain which contain one of the parts listed in pSB1A3 .

2. Inoculate two 3ml cultures of supplemented M9 Medium and antibiotic(Ampicillin 0.1mg/ml) with single colony from the plate.

3. Cultures were grown in test tubes（BIO BASIC INC.12ml Polypropylene Round-bottom Culture Tubes With Graduations And Dual Cap Cat.No:TD444） for 16hrs at 37℃ with shaking at 200rpm.

4. Cultures were diluted 1:1000 to tubes of 3ml fresh medium and grown for 4.5hrs.

5. Stock concentration of the cognate AHL, 3-oxohexanoyl-homoserine is diluted and added to different tubes to yield different final concentrations (1E-5,1E-7,1E-8,1E-9,1E-10M).To ensure the same response time , the AHL should be added with a time interval of 2mins between tubes, so do the measurements procedure.

6. Measure the fluorescence(SHIMDZU SPECTROFLUOROPHOTOMETER RF-5301PC, 250ul quartz cell path length 10mm,501 nm excitation,514 nm emission,1.5nm slit width) and absorbance ((HITACHI UV-VIS spectrophotometer U-2810 ,200ul quartz cell path length 10mm,600nm,1.5 nm slit width) for the first time 30 minutes after adding AHL. Repeat measurement every 30 mins in the next 4hrs.