Difference between revisions of "Part:BBa K176026"

Revision as of 07:56, 20 October 2009

TetR repressible AHL->GFP Receiver (Very High LuxR): pCon 0.70->luxR+pLux/Tet->GFP

AHL -> GFP

TetR repressible

Very high LuxR level

K176028 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 device.
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

T(hour)	OD600	FLU(arbitrary)	FLU/OD(arbitrary)	AHL(M)	ST.FLU/OD(ustc_st1)
0.5	0.152	0.513666667	3.379385965	0	13.50835818
0.5	0.164	0.547333333	3.337398374	1.00E-10	13.34052194
0.5	0.177	0.550666667	3.111111111	1.00E-09	12.43598797
0.5	0.146	0.442333333	3.029680365	1.00E-08	12.11048633
0.5	0.166	0.563666667	3.395582329	1.00E-07	13.57309961
0.5	0.167333333	0.541333333	3.235059761	1.00E-05	12.93144566
1.5	0.247333333	0.810666667	3.277628032	0	13.10160304
1.5	0.252	1.463	5.805555556	1.00E-10	23.20644184
1.5	0.254	15.52866667	61.13648294	1.00E-09	244.3797535
1.5	0.245	34.521	140.9020408	1.00E-08	563.2251701
1.5	0.261	35.881	137.4750958	1.00E-07	549.526705
1.5	0.254	39.50733333	155.5406824	1.00E-05	621.7399465
2.5	0.372	1.536666667	4.130824373	0	16.51206928
2.5	0.405333333	3.574666667	8.819078947	1.00E-10	35.2523442
2.5	0.411666667	68.232	165.745749	1.00E-09	662.5324739
2.5	0.403	177.9833333	441.6459884	1.00E-08	1765.383493
2.5	0.383666667	187.4133333	488.479583	1.00E-07	1952.59057
2.5	0.404	192.4866667	476.4521452	1.00E-05	1904.513512
3.5	0.722	3.111666667	4.309787627	0	17.22743585
3.5	0.873333333	8.359333333	9.571755725	1.00E-10	38.26100542
3.5	0.781	175.7666667	225.0533504	1.00E-09	899.6016725
3.5	0.734333333	373.0766667	508.0481162	1.00E-08	2030.811513
3.5	0.674333333	396.2633333	587.6371725	1.00E-07	2348.951403
3.5	0.72	400.7966667	556.662037	1.00E-05	2225.135056

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.

@@ Line 34: / Line 34: @@
 | align="center" style="background:#f0f0f0;"|'''FLU(arbitrary)'''
 | align="center" style="background:#f0f0f0;"|'''FLU/OD(arbitrary)'''
-| align="center" style="background:#f0f0f0;"|'''AHL'''
+| align="center" style="background:#f0f0f0;"|'''AHL(M)'''
 | align="center" style="background:#f0f0f0;"|'''ST.FLU/OD(ustc_st1)'''
 |-