Device

Part:BBa_K176217

Designed by: Danqian Liu, Zongxiao He, Hao Jiang   Group: iGEM09_USTC   (2009-10-08)

cell density->GFP: pCon 0.70->luxI-LVA+pCon 0.70->luxR+pLux/Tet->GFP

GFP = (Cell Density * pCon 0.70) + Input AHL

High level GFP inhibits cell growth.

This part is both a cell density reporter and a cell density controller.

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
    Illegal NheI site found at 856
    Illegal NheI site found at 879
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 698
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1834
    Illegal BsaI.rc site found at 2578


Model

To see the details of the model, click here please.


MEASUREMENT

We measured this device through two different methods. The first is examing the GFP expression at different time to test the intracellular AHL concentrations produced by K176020. We add different concentrations of AHL and then to test the response of this device to check it's response to both extracellular and intracellular AHL concentration. By combining two sets of data, we can build a modol to analyse the difference of AHL concentrations between extracellular and intracellular.

MEARSUMENT1

  • 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)
00.0875100.26051147.5570984587.109158401.3720513
600.120666667148.521229.3809034914.181969592.9779576
1200.145198.921370.076195476.580687794.1041996
1800.207666667292.45166671404.897875615.7727561166.208809

K176218


T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
00.103575.11366667721.40979242883.678268298.4607008
600.150333333123.0506667808.66590753232.465553485.9473215
1200.1995195.035966.4035763862.987472770.6660008
1800.308333333351.14833331131.8029784524.1355011394.941779

K176219

T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
00.251532.2705125.5594805501.8966322126.227003
600.37283333343.24566667112.4353648449.4358429167.5646634
1200.47483333368.484139.8281768558.9326329265.3998452
1800.594833333116.7865192.791143770.6405364458.4026791

K176220

T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
00.24857.72332.1438897128.488186931.92931443
600.410512.20229.72814969118.831793248.78045109
1200.49166666717.435536.20398381144.717527371.15278427
1800.61131.2646666751.84736322207.2485239126.6288481

K176221

T(min) OD600 FLU(arbitrary) FLU/OD600(arbitrary) ST.FLU/OD600(ustc_st1) ST.FLU(ustc_st1)
00.21966666711.4956666752.33304675209.189937845.95205634
600.349516.4136666746.97848701187.786253465.63129556
1200.45483333324.6943333354.29412657217.028926698.71199013
1800.576543.447575.41615552301.4596295173.7914764
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.


MEARSUMENT2

  • We then measured K176217 and K176219 again the same way with measurement1 but add different concentrations of AHL. Then, comparing with the data obtained by measurement1, we can build a model to analysis the difference of AHL concentrations between intracellular and extracellular.

data

K176217

AHL(m) T(h) OD600 FLU(arbitrary) FLU/OD600) ST.FLU/OD600(ustc_st1)
1.00E-100.50.07279.7281107.3333334425.792699
1.00E-090.50.07066666781.341151.0377364600.470567
1.00E-080.50.06833333384.726666671239.9024394955.64524
1.00E-070.50.07487.096333331176.9774774704.146593
1.00E-050.50.07233333385.2471178.5299544710.351534
1.00E-101.50.100333333109.551091.8604654363.9507
1.00E-091.50.099666667125.011254.2809365013.113255
1.00E-081.50.100333333123.081226.7109634902.921517
1.00E-071.50.099333333122.11333331229.3288594913.384728
1.00E-051.50.094333333109.21666671157.7738524627.393492
1.00E-102.50.129177.38666671375.0904395495.964985
1.00E-092.50.128666667181.051407.1243525623.998211
1.00E-082.50.122666667180.42666671470.8695655878.775241
1.00E-072.50.115666667170.01333331469.8559085874.723852
1.00E-052.50.101666667151.28666671488.0655745947.504292
1.00E-1040.171262.79666671536.8226126142.376547
1.00E-0940.171311.97666671824.4249517291.866312
1.00E-0840.146666667248.47333331694.1363646771.128552
1.00E-0740.130333333209.48666671607.3145786424.119017
1.00E-0540.109162.07333331486.9113155942.890947
 For the details of the definition of ST.FLU and the unit ustc_st1, please click here.

plot

Figure 1-the stereogram which shows the response of K176217 to both the additional AHL and time

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.



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Parameters
None