Part:BBa_K176136

aTc&AHL->GFP: pCon 0.70->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

K176136

AHL(M)	T(h)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)
0	0.5	0.191333333	0.230333333	1.203832753	4.811481825
1.00E-10	0.5	0.191	0.313666667	1.642233857	6.56368448
1.00E-09	0.5	0.193666667	0.412	2.127366609	8.502664306
1.00E-08	0.5	0.189333333	0.464333333	2.452464789	9.802017541
1.00E-07	0.5	0.188	0.483	2.569148936	10.26838104
1.00E-05	0.5	0.161666667	0.575	3.556701031	14.21543178
0	1.5	0.249	0.286666667	1.151271754	4.60140589
1.00E-10	1.5	0.249333333	0.411666667	1.651069519	6.598998876
1.00E-09	1.5	0.250666667	1.536	6.127659574	24.49104546
1.00E-08	1.5	0.248	2.437666667	9.829301075	39.28577568
1.00E-07	1.5	0.244	2.441666667	10.0068306	39.99532614
1.00E-05	1.5	0.202666667	2.552333333	12.59375	50.33473221
0	3	0.356666667	1.029333333	2.885981308	11.53469748
1.00E-10	3	0.361666667	1.441333333	3.985253456	15.92827121
1.00E-09	3	0.357333333	7.754333333	21.7005597	86.73285252
1.00E-08	3	0.356666667	11.61666667	32.57009346	130.1762328
1.00E-07	3	0.354	11.41833333	32.25517891	128.9175816
1.00E-05	3	0.297333333	11.67633333	39.27017937	156.9551534

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

plot

Figure 1- this steregram shows the response of K176136 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.