Difference between revisions of "Part:BBa K176217"

Revision as of 19:53, 20 October 2009

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

MEASUREMENT

*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

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

 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.