Part:BBa_K176127

TetR repressible AHL->PoPS Receiver (Medium LuxR): pCon 0.15->luxR+pLux/Tet->PoPS

AHL -> PoPS Receiver

TetR repressible

Medium 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

K176128

T(hour)	OD600	FLU(arbitrary)	FLU/OD(arbitrary)	AHL	ST.FLU/OD(ustc_st1)
0.5	0.144	0.805	5.590277778	0	22.34591589
0.5	0.156	1.197333333	7.675213675	1.00E-10	30.67999231
0.5	0.160333333	4.548333333	28.36798337	1.00E-09	113.394825
0.5	0.16	7.007	43.79375	1.00E-08	175.0559619
0.5	0.161333333	8.152333333	50.53099174	1.00E-07	201.986616
0.5	0.157333333	8.976666667	57.05508475	1.00E-05	228.0652546
1.5	0.256666667	1.568	6.109090909	0	24.4197582
1.5	0.274	1.970333333	7.190997567	1.00E-10	28.74444405
1.5	0.282666667	9.054	32.03066038	1.00E-09	128.0355773
1.5	0.276333333	64.781	234.4306393	1.00E-08	937.0853393
1.5	0.274333333	89.16466667	325.0230863	1.00E-07	1299.208883
1.5	0.264333333	83.70166667	316.6519546	1.00E-05	1265.74711
2.5	0.356	2.742333333	7.703183521	0	30.79179566
2.5	0.391666667	3.256666667	8.314893617	1.00E-10	33.23697333
2.5	0.421333333	17.24066667	40.9193038	1.00E-09	163.5659903
2.5	0.396666667	169.93	428.394958	1.00E-08	1712.415389
2.5	0.391666667	271.61	693.4723404	1.00E-07	2772.004399
2.5	0.371	247.67	667.574124	1.00E-05	2668.481928
3.5	0.471666667	4.409	9.34770318	0	37.36540425
3.5	0.499	5.094	10.20841683	1.00E-10	40.80591931
3.5	0.557333333	28.99833333	52.03050239	1.00E-09	207.9805828
3.5	0.518	282.62	545.5984556	1.00E-08	2180.910803
3.5	0.514333333	463.9933333	902.1257291	1.00E-07	3606.050802
3.5	0.483333333	470.7433333	973.9517241	1.00E-05	3893.159548

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.