Difference between revisions of "Part:BBa K176026"

Latest revision as of 02:01, 22 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

=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

Model

To see the details of the model, click [http://2009.igem.org/Team:USTC/Modeling/Model-2 here] please.

data

K176026

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.

Measurement2

K176026([[1]]) is a device which can response to different concentrations of AHL. Here, we use K176126 as a detector to examing the AHL concentrations produced by constitutive promoters + luxI.

constitutive promoters + luxI-LVA: 
                                                                K176020( J23101->luxI-LVA);
                                                                K176021( J23107->luxI-LVA);
                                                                K176022( J23115->luxI-LVA);
                                                                K176023( J23109->luxI-LVA);
                                                                K176024( J23103->luxI-LVA);

measuring device: 
                                    K176217:( K176020 + K176026):( K176083 + GFP); 
                                    K176218:( K176021 + K176026):( K176084 + GFP);
                                    K176219:( K176022 + K176026):( K176085 + GFP);
                                    K176220:( K176023 + K176026):( K176086 + GFP);
                                    K176221:( K176024 + K176026):( K176087 + GFP);

data

K176217

T(min)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)	ST.FLU(ustc_st1)
0	0.0875	100.2605	1147.557098	4587.109158	401.3720513
60	0.120666667	148.52	1229.380903	4914.181969	592.9779576
120	0.145	198.92	1370.07619	5476.580687	794.1041996
180	0.207666667	292.4516667	1404.89787	5615.772756	1166.208809

K176218

T(min)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)	ST.FLU(ustc_st1)
0	0.1035	75.11366667	721.4097924	2883.678268	298.4607008
60	0.150333333	123.0506667	808.6659075	3232.465553	485.9473215
120	0.1995	195.035	966.403576	3862.987472	770.6660008
180	0.308333333	351.1483333	1131.802978	4524.135501	1394.941779

K176219

T(min)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)	ST.FLU(ustc_st1)
0	0.2515	32.2705	125.5594805	501.8966322	126.227003
60	0.372833333	43.24566667	112.4353648	449.4358429	167.5646634
120	0.474833333	68.484	139.8281768	558.9326329	265.3998452
180	0.594833333	116.7865	192.791143	770.6405364	458.4026791

K176220

T(min)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)	ST.FLU(ustc_st1)
0	0.2485	7.723	32.1438897	128.4881869	31.92931443
60	0.4105	12.202	29.72814969	118.8317932	48.78045109
120	0.491666667	17.4355	36.20398381	144.7175273	71.15278427
180	0.611	31.26466667	51.84736322	207.2485239	126.6288481

K176221

T(min)	OD600	FLU(arbitrary)	FLU/OD600(arbitrary)	ST.FLU/OD600(ustc_st1)	ST.FLU(ustc_st1)
0	0.219666667	11.49566667	52.33304675	209.1899378	45.95205634
60	0.3495	16.41366667	46.97848701	187.7862534	65.63129556
120	0.454833333	24.69433333	54.29412657	217.0289266	98.71199013
180	0.5765	43.4475	75.41615552	301.4596295	173.7914764

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

plot

Figure 1- the relationship chart between FLU and OD600 of diffrent constitutive promoters + luxI-LVA

Figure 2- The time response of K176126 to different constitutive promoters + luxI-LVA. We can see from the figure that the more strong the constitutive promoter is , the more strong response it shows.

protocol

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 1:100 into 3ml fresh medium and grown for 3hrs.

5. 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) every 30 minutes in the next 4hrs.

@@ Line 5: / Line 5: @@
 TetR repressible
+Very high LuxR level
+[[Part:BBa_K176028|K176028]] with GFP.
 <!-- Add more about the biology of this part here
@@ Line 12: / Line 16: @@
 <span class='h3bb'>Sequence and Features</span>
 <partinfo>BBa_K176026 SequenceAndFeatures</partinfo>
+=='''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
+===Model===
+To see the details of the model, click [http://2009.igem.org/Team:USTC/Modeling/Model-2 here] please.
+===data===
+{|align = "center"
+|-
+|
+<font size = "3">K176026</font>
+|}
+{| {{table}}
+{|class="sortable" style="border:3px solid black; width: 70%" align="center"
+| align="center" style="background:#f0f0f0;"|'''T(hour)'''
+| align="center" style="background:#f0f0f0;"|'''OD600'''
+| align="center" style="background:#f0f0f0;"|'''FLU(arbitrary)'''
+| align="center" style="background:#f0f0f0;"|'''FLU/OD(arbitrary)'''
+| align="center" style="background:#f0f0f0;"|'''AHL(M)'''
+| align="center" style="background:#f0f0f0;"|'''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
+|}
+{{USTC//hybrid-AHL}}
+===Measurement2===
+{{USTC//K176217-1}}