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 [http://2009.igem.org/Team:USTC/Modeling/Model-5 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)
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.


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-10 0.5 0.072 79.728 1107.333333 4425.792699
1.00E-09 0.5 0.070666667 81.34 1151.037736 4600.470567
1.00E-08 0.5 0.068333333 84.72666667 1239.902439 4955.64524
1.00E-07 0.5 0.074 87.09633333 1176.977477 4704.146593
1.00E-05 0.5 0.072333333 85.247 1178.529954 4710.351534
1.00E-10 1.5 0.100333333 109.55 1091.860465 4363.9507
1.00E-09 1.5 0.099666667 125.01 1254.280936 5013.113255
1.00E-08 1.5 0.100333333 123.08 1226.710963 4902.921517
1.00E-07 1.5 0.099333333 122.1133333 1229.328859 4913.384728
1.00E-05 1.5 0.094333333 109.2166667 1157.773852 4627.393492
1.00E-10 2.5 0.129 177.3866667 1375.090439 5495.964985
1.00E-09 2.5 0.128666667 181.05 1407.124352 5623.998211
1.00E-08 2.5 0.122666667 180.4266667 1470.869565 5878.775241
1.00E-07 2.5 0.115666667 170.0133333 1469.855908 5874.723852
1.00E-05 2.5 0.101666667 151.2866667 1488.065574 5947.504292
1.00E-10 4 0.171 262.7966667 1536.822612 6142.376547
1.00E-09 4 0.171 311.9766667 1824.424951 7291.866312
1.00E-08 4 0.146666667 248.4733333 1694.136364 6771.128552
1.00E-07 4 0.130333333 209.4866667 1607.314578 6424.119017
1.00E-05 4 0.109 162.0733333 1486.911315 5942.890947 
 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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Categories
Parameters
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