Part:BBa_K176219
cell density->GFP: pCon 0.15->luxI-LVA+pCon 0.70->luxR+pLux/Tet->GFP
GFP = (Cell Density * pCon 0.15) + Input AHL
High level GFP inhibits cell growth.
This part is both a cell density reporter and a cell density controller.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE 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 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 698
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE 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
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
|
K176219 |
| AHL(m) | T(h) | OD600 | FLU(arbitrary) | FLU/OD600) | ST.FLU/OD600(ustc_st1) |
| 1.00E-10 | 0.5 | 0.141 | 28.94733333 | 205.3002364 | 820.54451 |
| 1.00E-09 | 0.5 | 0.150666667 | 31.041 | 206.0243363 | 823.4385943 |
| 1.00E-08 | 0.5 | 0.151666667 | 31.425 | 207.1978022 | 828.1287058 |
| 1.00E-07 | 0.5 | 0.147666667 | 31.86433333 | 215.785553 | 862.4522504 |
| 1.00E-05 | 0.5 | 0.156 | 36.18566667 | 231.9594017 | 927.0959301 |
| 1.00E-10 | 1.5 | 0.245333333 | 38.01633333 | 154.9578804 | 619.3360529 |
| 1.00E-09 | 1.5 | 0.253333333 | 99.74966667 | 393.7486842 | 1573.735748 |
| 1.00E-08 | 1.5 | 0.231 | 129.4333333 | 560.3174603 | 2239.478259 |
| 1.00E-07 | 1.5 | 0.221 | 122.0166667 | 552.1116139 | 2206.681111 |
| 1.00E-05 | 1.5 | 0.22 | 115.75 | 526.1363636 | 2102.863164 |
| 1.00E-10 | 2.5 | 0.391 | 59.505 | 152.1867008 | 608.2601949 |
| 1.00E-09 | 2.5 | 0.375666667 | 195.78 | 521.1535049 | 2082.947661 |
| 1.00E-08 | 2.5 | 0.3 | 245.83 | 819.4333333 | 3275.113243 |
| 1.00E-07 | 2.5 | 0.259 | 209.82 | 810.1158301 | 3237.873022 |
| 1.00E-05 | 2.5 | 0.252 | 180.5733333 | 716.5608466 | 2863.952224 |
| 1.00E-10 | 4 | 0.514 | 98.96333333 | 192.535668 | 769.5270502 |
| 1.00E-09 | 4 | 0.537 | 378.7266667 | 705.2638113 | 2818.800205 |
| 1.00E-08 | 4 | 0.402 | 461.4933333 | 1147.993367 | 4588.302824 |
| 1.00E-07 | 4 | 0.319666667 | 330.06 | 1032.513034 | 4126.750737 |
| 1.00E-05 | 4 | 0.284666667 | 242.3333333 | 851.2880562 | 3402.430281 |
For the details of the definition of ST.FLU and the unit ustc_st1, please click here.
plot
protocol
AHLHybrid 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.
| None |