Part:BBa_K176125

TetR repressible AHL->PoPS Receiver (High LuxR): pCon 0.36->luxR+pLux/Tet->PoPS

AHL -> PoPS Receiver

TetR repressible

High LuxR level

Similar to F2620, but use pLux/Tet hybrid promoter.

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

MEASUREMENT

We mesured the hybrid promoter's response to AHL concentration through different devices.
INPUT: AHL
DEVICE: 
             TetR repressible AHL->GFP Receiver:
              K176028 ( pCon 0.70->luxR+pLux/Tet)->GFP ;
             K176125 ( pCon 0.36->luxR+pLux/Tet)->GFP ;
             K176127 ( pCon 0.15->luxR+pLux/Tet)->GFP ;
             K176129 (pCon 0.04->luxR+pLux/Tet)->GFP ;
OUTPUT: GFP

data

K176126

T(hour)	OD600	FLU(arbitrary)	FLU/OD(arbitrary)	AHL	ST.FLU/OD(ustc_st1)
0.5	0.147	0.724	4.925170068	0	19.68729291
0.5	0.149666667	2.844	19.00222717	1.00E-10	75.95725775
0.5	0.157	9.368666667	59.67303609	1.00E-09	238.529944
0.5	0.152	11.27633333	74.18640351	1.00E-08	296.5439641
0.5	0.149333333	11.57933333	77.54017857	1.00E-07	309.9499483
0.5	0.147	12.41233333	84.43764172	1.00E-05	337.5210526
1.5	0.268	1.283666667	4.789800995	0	19.14618457
1.5	0.276666667	3.247666667	11.73855422	1.00E-10	46.9223097
1.5	0.282666667	38.829	137.3667453	1.00E-09	549.0935975
1.5	0.270333333	94.57733333	349.8545006	1.00E-08	1398.467045
1.5	0.255666667	92.93366667	363.4954368	1.00E-07	1452.993711
1.5	0.232	75.93966667	327.3261494	1.00E-05	1308.414876
2.5	0.381666667	2.555	6.694323144	0	26.75909639
2.5	0.439	5.467	12.45330296	1.00E-10	49.77936188
2.5	0.437333333	92.855	212.3208841	1.00E-09	848.7064162
2.5	0.409333333	227.38	555.4885993	1.00E-08	2220.444495
2.5	0.382	222.79	583.2198953	1.00E-07	2331.294301
2.5	0.299333333	163.65	546.714922	1.00E-05	2185.373634
3.5	0.584	4.336	7.424657534	0	29.67844879
3.5	0.611	7.840333333	12.83196945	1.00E-10	51.29299856
3.5	0.599333333	154.5333333	257.8420467	1.00E-09	1030.667333
3.5	0.574333333	412.9266667	718.9669182	1.00E-08	2873.913412
3.5	0.518333333	401.8466667	775.266881	1.00E-07	3098.960231
3.5	0.381666667	272.7966667	714.7510917	1.00E-05	2857.061565

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.