Part:BBa_K176140

aTc&AHL->GFP: pCon 0.15->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

K176140

AHL(M)	T(h)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)
0	0.5	0.236666667	2.070666667	8.749295775	34.96920773
1.00E-10	0.5	0.237666667	1.967	8.276297335	33.07872636
1.00E-09	0.5	0.246	4.862666667	19.76693767	79.00454704
1.00E-08	0.5	0.242666667	4.767	19.64423077	78.51411179
1.00E-07	0.5	0.237666667	5.182	21.80364656	87.14487036
1.00E-05	0.5	0.24	5.683	23.67916667	94.6409539
0	1.5	0.417666667	2.472	5.918595371	23.65545712
1.00E-10	1.5	0.415	3.980666667	9.591967871	38.33720172
1.00E-09	1.5	0.413333333	149.79	362.3951613	1448.421908
1.00E-08	1.5	0.405333333	132.8333333	327.7138158	1309.807417
1.00E-07	1.5	0.402	136.3766667	339.2454395	1355.89704
1.00E-05	1.5	0.396	134.04	338.4848485	1352.857108
0	3	0.616	6.812	11.05844156	44.19840751
1.00E-10	3	0.651666667	11.56666667	17.74936061	70.9406899
1.00E-09	3	0.616	635.81	1032.159091	4125.336095
1.00E-08	3	0.608	646.3333333	1063.048246	4248.793947
1.00E-07	3	0.605	655.2066667	1082.986226	4328.482118
1.00E-05	3	0.594	696.75	1172.979798	4688.168657

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

plot

Figure 2- this steregram shows the response of K176138 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.