Part:BBa_K176126

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

AHL -> GFP Receiver

TetR repressible

High LuxR level

K176125 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

MEASUREMENT1

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

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.

MEARSUREMENT2

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