Difference between revisions of "Part:BBa K201001:Experience"

Latest revision as of 01:31, 22 October 2009

Dh5alpha cells were co-transformed with the BBa_K201001 on a high copy number plasmid (pSB1A2) and BBa_K201002 on a low copy number plasmid (pSB3K3). To characterize this device and its sensitivity to the inducer, we studied both the static and the dynamic response to IPTG induction.

Static response Dh5alpha were inoculated in 5 ml of M9 medium with 0, 10, 20, 40, 60, 80, 100 uM IPTG, respectively. After O/N growth at 37° (about 12 h) samples were collected and slides prepared for microscope analysis. Acquired images were analyzed with the [http://2009.igem.org/Team:Bologna/Software VIFluoR software]. To obtain a significant representation of bacterial fluorescence, it was necessary to acquire several images, each one reporting a sufficient number of bacterial cells (n=60). VIFluoR operates image segmentation and then recognises the bacterial cells, yielding the mean fluorescence per bacterium as the output. The experimental data (Fig. 1) were used to identify, by the [http://2009.igem.org/Team:Bologna/Modeling mathematical model], the operator binding affinity for the repressor LacI (K= 1.7 nM).

Fig.1. Experimental data (blue lines) of the static induction after 0, 10, 20, 40, 60, 80, 100 uM IPTG induction. Data were fitted by the model (green line) to identify the operator-repressor binding affinity (K= 1.7 nM)

After parameter identification, we computed by the model the static control curve for the LacI repressed GFP generator (LacI inverter) (Fig.2).

Fig.2. Model prediction of promoter repression by Lac I.

Dynamic response Dh5alpha cells were inoculated in the morning (9 a.m.) in 5 ml of M9 medium with no IPTG. After daily growth (about 8 h) the culture was diluted to an OD=0.1. To perform the induction analysis, the culture was splitted in two. A half was induced with 100 uM IPTG and the other was grown in control medium. 200 ul of each sample were used to fill plate wells (6 wells each). Cells were grown into a fluorimeter (Tecan M200) O/N (about 12h) at 37°. OD and fluorescence were sampled every 15 min (Fig. 3 and 4, respectively).

Fig.3. Growth curve for the uninduced (black line) and induced (100 uM IPTG, light blue line)system.

Fig.4. Absolute fluorescence curve for the uninduced (black line) and induced (light blue, 100 uM IPTG)system.

Fig.5. Model fitting of the experimental data. Experimental data (black lines) were fitted by the model considering a constant (blue line) or a varying (green line) amount of RNA polymerase

Experimental data of the fluorescence/OD ratio (Fig. 5; blue symbols) were compared to model predictions obtained either considering a constant (purple line) or progressively increasing (green line) amount of RNA polymerase. A good fitting can only be obtained if RNA polymerase available for transcription increases up to 30fold with respect to the initial value. This is consistent with the required activation of the major sigma subunit of RNA polymerase for transcription of most of the genes expressed in the exponential growth phase (Jishage M, Ishihama A. Proc Natl Acad Sci USA 1998; 95: 4953–8).

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-To experimentally and mathematically characterize the device and its sensitive to the inducer, we studied both the static and a dynamic response to  IPTG induction.
+<div style="text-align:justify">
+Dh5alpha cells were co-transformed with the BBa_K201001 on a high copy number plasmid (pSB1A2) and [https://parts.igem.org/Part:BBa_K201002 BBa_K201002] on a low copy number plasmid (pSB3K3).  To characterize this device and its sensitivity to the inducer, we studied both the static and the dynamic response to IPTG induction.
-Dh5alpha cells were co-transformed with the BBa_K201001 on a high copy number plasmid (pSB1A2) and [https://parts.igem.org/Part:BBa_K201002 BBa_K201002] on a low copy number plasmid (pSB3K3).
 '''Static response'''
-Dh5alpha were inoculated in 5 ml of M9 medium with 0, 10, 20, 40, 60, 80, 100 uM IPTG, respectively.  After O/N growth at 37° (about 12 h) samples were collected and slides prepared for microscope analysis.  Images were then analyzed with the [http://2009.igem.org/Team:Bologna/Software VIFluoR software]. To obtain a significant representation of bacterium fluorescence, it was necessary to acquire several images, each one reporting a sufficient number of bacterial cells (n=60). VIFluoR operates the image segmentation and then recognises the bacterial cells yielding the mean fluorescence per bacterium as the output. The experimental data (Fig. 1) were used to identify, by the [http://2009.igem.org/Team:Bologna/Modeling mathematical model], the operator binding affinity for the repressor LacI '''(K= 1.7 nM)'''.
+Dh5alpha were inoculated in 5 ml of M9 medium with 0, 10, 20, 40, 60, 80, 100 uM IPTG, respectively.  After O/N growth at 37° (about 12 h) samples were collected and slides prepared for microscope analysis. Acquired images were analyzed with the [http://2009.igem.org/Team:Bologna/Software VIFluoR software]. To obtain a significant representation of bacterial fluorescence, it was necessary to acquire several images, each one reporting a sufficient number of bacterial cells (n=60). VIFluoR operates image segmentation and then recognises the bacterial cells, yielding the mean fluorescence per bacterium as the output. The experimental data (Fig. 1) were used to identify, by the [http://2009.igem.org/Team:Bologna/Modeling mathematical model], the operator binding affinity for the repressor LacI '''(K= 1.7 nM)'''.
 <br>
 [[Image:static_induction_figure.jpg|center|600px|thumb|Fig.1. Experimental data (blue lines) of the static induction after 0, 10, 20, 40, 60, 80, 100 uM IPTG induction. Data were fitted by the model (green line) to identify the operator-repressor binding affinity ('''K= 1.7 nM''')]]
-After parameter identification, we computed by the model the static control curve  for the LacI repressed GFP generator (LacI inverter) (Fig.2).
+After parameter identification, we computed by the model the static control curve for the LacI repressed GFP generator (LacI inverter) (Fig.2).
+[[Image:LacI_GFP.jpg|center|600px|thumb|Fig.2.  Model prediction of promoter repression by Lac I.]]
 '''Dynamic response'''
-Dh5alpha cells were inoculated in the morning (9 a.m.) in 5 ml of M9 medium with no IPTG. After daily growth (about 8 h) the culture was diluited to an OD=0.1. To perform the induction analysis, the culture was splitted in two. A half was induced with 100 uM IPTG and the other was grown in control medium. 200 ul of each sample were used to fill plate wells (6 wells each). Cells were grown into a fluorimeter (Tecan M200) O/N (about 12h) at 37°. OD and fluorescence were sampled each 15 min (Fig. 3 and 4, respectively).
+Dh5alpha cells were inoculated in the morning (9 a.m.) in 5 ml of M9 medium with no IPTG. After daily growth (about 8 h) the culture was diluted to an OD=0.1. To perform the induction analysis, the culture was splitted in two. A half was induced with 100 uM IPTG and the other was grown in control medium. 200 ul of each sample were used to fill plate wells (6 wells each). Cells were grown into a fluorimeter (Tecan M200) O/N (about 12h) at 37°. OD and fluorescence were sampled every 15 min (Fig. 3 and 4, respectively).
-[[Image:OD.png|center|600px|thumb|Fig.3. Growth curve for the uninduced (black line) and induced (100 uM IPTG, red line)system.]]
-[[Image:fluorescenza.png|center|600px|thumb|Fig.4. Absolute fluorescence curve for the uninduced (black line) and induced (red line, 100 uM IPTG)system.]]
-[[Image:induction_figure.jpg|center|600px|thumb|Fig.5. Model fitting of the experimental data. Experimental data (blue lines) were fitted by the model considering a constant (red line) or a varying (green line) amount of RNA polymerase]]
-Fluorescence over OD was compared with  the model prediction (Fig. 5) considering both a constant and a varying amount of the RNA polymerase enzyme. A good fitting can only be obtained if a varying Polymerase is consider. This can be explained considering the GFP expression  depending on the growth phase.
-Fig. 6 shows LacI promoter repression as predicted by the model simulation.
+[[Image:OD1.png|center|600px|thumb|Fig.3. Growth curve for the uninduced (black line) and induced (100 uM IPTG, light blue line)system.]]
+[[Image:Fluorescenza1.png|center|600px|thumb|Fig.4. Absolute fluorescence curve for the uninduced (black line) and induced (light blue, 100 uM IPTG)system.]]
+[[Image:induction_figure.jpg|center|600px|thumb|Fig.5. Model fitting of the experimental data. Experimental data (black lines) were fitted by the model considering a constant (blue line) or a varying (green line) amount of RNA polymerase]]
-[[Image:LacI_GFP.jpg|center|600px|thumb|Fig.6. LacI promoter repression as predicted by the model.]]
+Experimental data of the fluorescence/OD ratio (Fig. 5; blue symbols) were compared to model predictions obtained either considering a constant (purple line) or progressively increasing (green line) amount of RNA polymerase. A good fitting can only be obtained if RNA polymerase available for transcription increases up to 30fold with respect to the initial value. This is consistent with the required activation of the major sigma subunit of RNA polymerase for transcription of most of the genes expressed in the exponential growth phase (Jishage M, Ishihama A. Proc Natl Acad Sci USA 1998; 95: 4953–8).
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