Part:BBa_K176130

TetR repressible AHL->GFP Receiver (Low LuxR): pCon 0.04->luxR+pLux/Tet->GFP

AHL -> GFP Receiver

TetR repressible

Low LuxR level

K176129 with 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
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 985
Illegal BsaI.rc site found at 1729

MEASUREMENT

We mesured the hybrid promoter's response to AHL concentration through different devices.
INPUT: AHL
DEVICE: 
             TetR repressible AHL->GFP Receiver:
             K176026 ( pCon 0.70->luxR+pLux/Tet->GFP );
             K176126 ( pCon 0.36->luxR+pLux/Tet->GFP );
             K176128 ( pCon 0.15->luxR+pLux/Tet->GFP );
             K176130 (pCon 0.04->luxR+pLux/Tet->GFP );
OUTPUT: GFP

data

K176130

T(hour)	OD600	FLU(arbitrary)	FLU/OD(arbitrary)	AHL	ST.FLU/OD(ustc_st1)
0.5	0.140666667	0.832666667	5.91943128	0	23.66163521
0.5	0.146666667	0.747666667	5.097727273	1.00E-10	20.3770527
0.5	0.151	1.635	10.82781457	1.00E-09	43.28182664
0.5	0.144666667	5.035333333	34.80645161	1.00E-08	139.1311972
0.5	0.145	6.813666667	46.9908046	1.00E-07	187.8354903
0.5	0.147	7.474	50.84353741	1.00E-05	203.2359492
1.5	0.249666667	1.616333333	6.473965287	0	25.87826393
1.5	0.262	1.293666667	4.937659033	1.00E-10	19.73721483
1.5	0.266333333	1.912666667	7.181476846	1.00E-09	28.70638704
1.5	0.256	9.243	36.10546875	1.00E-08	144.3237349
1.5	0.248666667	26.90566667	108.1997319	1.00E-07	432.5048243
1.5	0.243	52.47433333	215.9437586	1.00E-05	863.1880664
2.5	0.407	3.353666667	8.23996724	0	32.93747148
2.5	0.414	2.235666667	5.400161031	1.00E-10	21.58596567
2.5	0.424	3.147333333	7.422955975	1.00E-09	29.67164718
2.5	0.41	15.49433333	37.79105691	1.00E-08	151.0615058
2.5	0.394333333	55.61766667	141.0422654	1.00E-07	563.7856874
2.5	0.39	139.1066667	356.6837607	1.00E-05	1425.765522
3.5	0.565666667	9.078	16.04832057	0	64.14966049
3.5	0.577333333	3.753	6.500577367	1.00E-10	25.98463991
3.5	0.607	5.145333333	8.476661175	1.00E-09	33.88360385
3.5	0.579666667	26.34833333	45.45428407	1.00E-08	181.6935846
3.5	0.557666667	98.57133333	176.7567244	1.00E-07	706.5464462
3.5	0.558	267.6666667	479.6893668	1.00E-05	1917.453599

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

plot

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.