From 2008.igem.org

(Difference between revisions)

Latest revision as of 05:49, 27 October 2008

Characterization of the existing pLacI part (BBa_R0010)

For the protocol of the characterization experiment, please click on the file: File:NTU@iGEM Characterization Protocol.pdf
Link to the registry for Biobrick part:[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 BBa_R0010]

pLacI promoter with GFP reporting gene

Results obtained at 25°C (Graph 4-1)

Characterization of pLaci-GFP at 25°C

Observations
From the graph 4-1 of RFU vs. Lactose Concentration for the experiment conducted at 25°C, we could deduce the following:

1) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings. This could be due the cells accommodating to an environment that was of a different medium and temperature. As previously, the cells were incubated at 37°C and in LB medium, but during the RFU measurement, the temperature was 25° and M9 medium was used instead.

2) After about 90 minutes, the RFU readings start to increase. The cells most likely have become accustomed to the new environment. The effects of the Lactose inoculation took place, activating the pLacI promoter and thus producing GFP proteins which led to a higher RFU measurement.

3) An interesting observation was made. As more readings were taken over time, the effect of Lactose concentration was more significant for cells that were subjected to lower Lactose concentration. As shown in Graph 4-1, we could see that the RFU readings for lower concentration of Lactose were higher than that for the cells inoculated with a higher concentration of Lactose.

4) After approximately 400 minutes, the RFU readings generally become constant with slight fluctuations observed. This could be due to the cells reaching saturation where the lactose in the environment would not further increase the amount of GFP protein formed. Another reason could be due the cell reaching stationary phrase, where the cells would not be affected by the lactose in their environment.

Results obtained at 37°C (Graph 4-2)

Characterization of pLaci-GFP at 37°C

Observations
From the graph 4-2 of RFU vs. Lactose Concentration for the experiment conducted at 37°C, we could deduce the following:

1) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings. This could be due the cells accommodating to a environment that was of a different medium. Previously the cell was incubated in LB medium. In this experiment M9 medium was used instead as it would not auto-fluoresce.

2) After about 90 minutes, the RFU readings start to increase. A similar observation was made for the cells that were subjected to a temperature of 25°C in the previous experiment.

3) As more readings were taken over time, the effect of Lactose concentration was more prominent for the cells that were subjected to higher Lactose inoculation. As shown in Graph 4-2, we could see that the RFU readings for higher concentration of Lactose were higher than that for lower concentration of Lactose. This result shows that an increase in the amount of lactose would cause an increase in LacI promoter activity. This is proven by an increase in RFU readings. This result was unlike those observed for the experiment done at 25°C.

4) After approximately 400 minutes, the RFU readings generally become constant with slight fluctuations observed, similar to the results found in 25°C (Graph 4-1).

Verification

The above observations could also be simply due to the multiplication of cells over time, hence with an increase in cell number, there would be also an increase in basal amount of GFP produced. Furthermore, with an increase in lactose concentration, the cells would have more nutrients for its growth.

In order to investigate this further, the fluorescence microscope pictures shown below were taken for the varying concentration of lactose. From the diagram below, it could be seen that the E Coli cells at 10mM lactose concentration fluoresce brighter than those at 1mM lactose concentration. This shows that the effect of increasing the lactose concentration resulted in more GFP formed and hence a higher RFU reading.

Fluorescence Microscope picture of pLacI-GFP at 1.0mM Lactose concentration

Fluorescence Microscope picture of pLacI-GFP at 10.0mM Lactose concentration

Results obtained at 42°C (Graph 4-3)

Characterization of pLaci-GFP at 42°C

Observations From the graph 4-3 of RFU vs. Lactose Concentration for the experiment conducted at 42°C, we could deduce the following:

1) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings.

2) The normalized RFU readings for 42°C (ranging from -15 to 40) were generally lower than those obtained for 25°C (ranging from -50 to 150) and for 37°C (ranging from 0 to 500). This small range increased could be due to the relatively low activity when the temperature was at 42°C. The low activity showed that at 42°C, the condition was not conducive for the cells to express the GFP protein.

3) After about 50 minutes, the RFU readings start to increase for all concentration of Lactose.

4) As more readings were taken over time, the effect of Lactose concentration was not significantly more for cells that were subjected to higher Lactose concentration. As shown in Graph 4-3, we could see that the RFU readings for higher concentration of Lactose only deviates from those readings for lower concentration of Lactose by less than 10%. This was different from the result observed when the cells were inoculated at 37°C, suggesting that the temperature does play an important role on how the cells may respond.

5) After approximately 400 minutes, the RFU readings showed trends of increasing, though the increase was minimal. This was not similar to the results obtained in 25°C (Graph 4-1) and 37°C (Graph 4-2). Again the temperature could be the main reason why the cells are behaving in this manner.

Measurement compared to Standard Promoters

RFU Measurements After 3 hours

For Lactose induction of 0 to 10 mM

Characterization Graph of LacI-GFP after 3 hours

A comparision of the Standards promoters with pLacI

Characterization Graph of Standards & LacI-GFP after 3 hours

In comparison with the graphs provided in the Measurement Kit

Practice promoter set included with the measurement kit to practice characterization.

RFU Measurements After 3.5 hours

For Lactose induction of 0 to 10 mM

Characterization Graph of LacI-GFP after 3.5 hours

A comparision of the Standards promoters with pLacI

Characterization Graph of Standards & LacI-GFP after 3.5 hours

The detailed data points and graphs shown above can be retrieved from the File:NTU@iGEM Results for characterization pLacI 010708.xls.

@@ Line 6: / Line 6: @@
-=Characterization of the existing LacI-GFP part (BBa_R0010)=
+='''Characterization of the existing pLacI part (BBa_R0010)'''=
-==Results obtained at 25°C (Graph 4-1)==
+For the protocol of the characterization experiment, please click on the file: [[Image:NTU@iGEM Characterization Protocol.pdf]]<br>
-[[Image:NTU@IGEM_LacI-GFP_25_deg_normal.JPG|900px]]
+Link to the registry for Biobrick part:[http://partsregistry.org/wiki/index.php?title=Part:BBa_R0010 BBa_R0010]
+[[Image:NTU@iGEM_PLacI_GFP_pic.JPG|center|thumb|400px|pLacI promoter with GFP reporting gene]]
+==''Results obtained at 25°C (Graph 4-1)''==
+[[Image:NTU@IGEM_LacI-GFP_25_deg_normal.JPG|900px|thumb|center|Characterization of pLaci-GFP at 25°C]]
+'''Observations'''<br>
 From the graph 4-1 of RFU vs. Lactose Concentration for the experiment conducted at 25°C, we could deduce the following:
-) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings. This could be due the cells accommodating to a environment that was of a different medium and temperature. As previously, the cells were incubated at 37°C and in LB medium, but during the RFU measurement, the temperature was 42° and M9 medium was used instead as it would not auto-fluoresce.
+) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings. This could be due the cells accommodating to an environment that was of a different medium and temperature. As previously, the cells were incubated at 37°C and in LB medium, but during the RFU measurement, the temperature was 25° and M9 medium was used instead.
-) After about 90 minutes, the RFU readings start to increase for all concentration of Lactose. This could be due to the fact that the cells were fully accommodated, and were starting to show the effects of the Lactose, the activator of the LacI-GFP promoter, thus producing GFP proteins which led to more RFU measured.
+) After about 90 minutes, the RFU readings start to increase. The cells most likely have become accustomed to the new environment. The effects of the Lactose inoculation took place, activating the pLacI promoter and thus producing GFP proteins which led to a higher RFU measurement.
-) A strange observation was observed, as more readings were taken over time, the effect of Lactose concentration was more on cells that were subjected to lower Lactose concentration. As shown in Graph 4-1, we could see that the RFU readings for lower concentration of Lactose were higher than that for higher concentration of Lactose. This puzzling result could be due to the cells being overwhelmed by the high amounts of Lactose.
+) An interesting observation was made. As more readings were taken over time, the effect of Lactose concentration was more significant for cells that were subjected to lower Lactose concentration. As shown in Graph 4-1, we could see that the RFU readings for lower concentration of Lactose were higher than that for the cells inoculated with a higher concentration of Lactose.
-) After approximately 400 minutes, the RFU readings generally become constant with slight fluctuations observed. This could be due the cells reaching saturation where the lactose in the environment would not further increase the amount of GFP protein formed. This could also be due the cell reaching stationary phrase, where the cells would not be actively affected by the lactose in the environment.
+) After approximately 400 minutes, the RFU readings generally become constant with slight fluctuations observed. This could be due to the cells reaching saturation where the lactose in the environment would not further increase the amount of GFP protein formed. Another reason could be due the cell reaching stationary phrase, where the cells would not be affected by the lactose in their environment.
-==Results obtained at 37°C (Graph 4-2)==
+==''Results obtained at 37°C (Graph 4-2)''==
-[[Image:NTU@IGEM_LacI-GFP_37_deg_normal.JPG|900px]]
+[[Image:NTU@IGEM_LacI-GFP_37_deg_normal.JPG|thumb|center|900px|Characterization of pLaci-GFP at 37°C]]
+'''Observations'''<br>
 From the graph 4-2 of RFU vs. Lactose Concentration for the experiment conducted at 37°C, we could deduce the following:
-) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings. This could be due the cells accommodating to a environment that was of a different medium. As previously, the cells were incubated in LB medium, but during the RFU measurement, M9 medium was used instead as it would not auto-fluoresce.
+) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings. This could be due the cells accommodating to a environment that was of a different medium. Previously the cell was incubated in LB medium. In this experiment M9 medium was used instead as it would not auto-fluoresce.
-) After about 90 minutes, the RFU readings start to increase for all concentration of Lactose. This could be due to the fact that the cells were fully accommodated, and were starting to show the effects of the Lactose, the activator of the LacI promoter, thus producing GFP proteins which led to more RFU measured.
+) After about 90 minutes, the RFU readings start to increase. A similar observation was made for the cells that were subjected to a temperature of 25°C in the previous experiment.
-) As more readings were taken over time, the effect of Lactose concentration was more on cells that were subjected to higher Lactose concentration. As shown in Graph 4-2, we could see that the RFU readings for higher concentration of Lactose were higher than that for lower concentration of Lactose. This result could be due the increase in amount of lactose directly caused an increase in LacI promoter activity, which further results in more GFP protein formed and an increase in RFU readings measured.
+) As more readings were taken over time, the effect of Lactose concentration was more prominent for the cells that were subjected to higher Lactose inoculation. As shown in Graph 4-2, we could see that the RFU readings for higher concentration of Lactose were higher than that for lower concentration of Lactose. This result shows that an increase in the amount of lactose would cause an increase in LacI promoter activity. This is proven by an increase in RFU readings. This result was unlike those observed for the experiment done at 25°C.
-) After approximately 400 minutes, the RFU readings generally become constant with slight fluctuations observed, similar to the results found in 25°C (Graph 4-1). This could be due the cells reaching saturation, where lactose in the environment would not further increase the amount of GFP protein formed. This could also be due the cell reaching stationary phrase, where the cells would not be actively affected by the lactose in the environment.
+) After approximately 400 minutes, the RFU readings generally become constant with slight fluctuations observed, similar to the results found in 25°C (Graph 4-1).
+==='''Verification'''===
+The above observations could also be simply due to the multiplication of cells over time, hence with an increase in cell number, there would be also an increase in basal amount of GFP produced. Furthermore, with an increase in lactose concentration, the cells would have more nutrients for its growth.
+In order to investigate this further, the fluorescence microscope pictures shown below were taken for the varying concentration of lactose. From the diagram below, it could be seen that the E Coli cells at 10mM lactose concentration fluoresce brighter than those at 1mM lactose concentration. This shows that the effect of increasing the lactose concentration resulted in more GFP formed and hence a higher RFU reading.
+[[Image:NTU@iGEM_Fluorscence_LacI-GFP_1mM.JPG|thumb|center|500px|Fluorescence Microscope picture of pLacI-GFP at 1.0mM Lactose concentration]]
+[[Image:NTU@iGEM_Fluorscence_LacI-GFP_10mM.JPG|thumb|center|500px|Fluorescence Microscope picture of pLacI-GFP at 10.0mM Lactose concentration]]
 ==Results obtained at 42°C (Graph 4-3)==
-[[Image:NTU@IGEM_LacI-GFP_42_deg_normal.JPG.jpg|900px]]
+[[Image:NTU@IGEM_LacI-GFP_42_deg_normal.JPG.jpg|thumb|center|900px|Characterization of pLaci-GFP at 42°C]]
+'''Observations'''
 From the graph 4-3 of RFU vs. Lactose Concentration for the experiment conducted at 42°C, we could deduce the following:
-) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings. This could be due the cells accommodating to a environment that was of a different medium and temperature. As previously, the cells were incubated at 37°C and in LB medium, but during the RFU measurement, the temperature was 42° and M9 medium was used instead as it would not auto-fluoresce.
+) For all concentration of Lactose (0 to 10mM), there were initial dips in RFU readings.
 ) The normalized RFU readings for 42°C (ranging from -15 to 40) were generally lower than those obtained for 25°C (ranging from -50 to 150) and for 37°C (ranging from 0 to 500). This small range increased could be due to the relatively low activity when the temperature was at 42°C. The low activity showed that at 42°C, the condition was not conducive for the cells to express the GFP protein.
-) After about 50 minutes, the RFU readings start to increase for all concentration of Lactose. This could be due to the fact that the cells had started to accommodate, and were starting to show the effects of the Lactose, the activator of the LacI promoter, thus producing GFP proteins which led to more RFU measured.
+) After about 50 minutes, the RFU readings start to increase for all concentration of Lactose.
-) As more readings were taken over time, the effect of Lactose concentration was NOT significantly more for cells that were subjected to higher Lactose concentration. As shown in Graph 4-3, we could see that the RFU readings for higher concentration of Lactose were about the same to those readings for lower concentration of Lactose. This result could be due the increase in amount of lactose which directly caused an increase in LacI promoter activity. However the effect of temperature was more dominant, as only a small increase of RFU readings were measured.
+) As more readings were taken over time, the effect of Lactose concentration was not significantly more for cells that were subjected to higher Lactose concentration. As shown in Graph 4-3, we could see that the RFU readings for higher concentration of Lactose only deviates from those readings for lower concentration of Lactose by less than 10%. This was different from the result observed when the cells were inoculated at 37°C, suggesting that the temperature does play an important role on how the cells may respond.
-) After approximately 400 minutes, the RFU readings showed trends of increasing, though the increase was minimal. This was not similar to the results obtained in 25°C (Graph 4-1) and 37°C (Graph 4-2). This could be due the cells not reaching saturation yet and the lactose in the environment caused the increased in amount of GFP protein formed.
+) After approximately 400 minutes, the RFU readings showed trends of increasing, though the increase was minimal. This was not similar to the results obtained in 25°C (Graph 4-1) and 37°C (Graph 4-2). Again the temperature could be the main reason why the cells are behaving in this manner.
-==Measurement compared to Standard Promoters==
+=='''Measurement compared to Standard Promoters'''==
-===RFU Measurements After 3 hours===
+==='''RFU Measurements After 3 hours'''===
 ====For Lactose induction of 0 to 10 mM====
 [[Image:NTU@iGEM LacI-GFP RFU Chart 3 hours.JPG|thumb|center|700px|Characterization Graph of LacI-GFP after 3 hours]]
-====In comparision with the Standards====
+====A comparision of the Standards promoters with pLacI====
 [[Image:NTU@iGEM_Standard_promoter_with_LacI-GFP_RFU_Chart_3_hours.JPG|thumb|center|700px|Characterization Graph of Standards & LacI-GFP after 3 hours]]
-===RFU Measurements After 3.5 hours===
+===In comparison with the graphs provided in the Measurement Kit===
+[[Image:NTU@iGEM_Promoter_Practice_Set.png|thumb|center|900px|Practice promoter set included with the measurement kit to practice characterization.]]
+==='''RFU Measurements After 3.5 hours'''===
 ====For Lactose induction of 0 to 10 mM====
-[[Image:NTU_Characterization_Graph_LacI_GFPs_(3.5_hours).JPG|thumb|center|700px|Characterization Graph of LacI-GFP after 3.5 hours]]
+[[Image:NTU@iGEM_LacI-GFP_RFU_Chart_3.5_hours.JPG|thumb|center|700px|Characterization Graph of LacI-GFP after 3.5 hours]]
+====A comparision of the Standards promoters with pLacI====
+[[Image:NTU@iGEM_Standard_promoter_with_LacI-GFP_RFU_Chart_3.5_hours.JPG|thumb|center|700px|Characterization Graph of Standards & LacI-GFP after 3.5 hours]]
+The detailed data points and graphs shown above can be retrieved from the [[image: NTU@iGEM_Results_for_characterization_pLacI_010708.xls]].
+<br><br>
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Team:NTU-Singapore/Parts/Characterization of LacI-GFP

From 2008.igem.org

Latest revision as of 05:49, 27 October 2008

Contents

Characterization of the existing pLacI part (BBa_R0010)

Results obtained at 25°C (Graph 4-1)

Results obtained at 37°C (Graph 4-2)

Verification

Results obtained at 42°C (Graph 4-3)

Measurement compared to Standard Promoters

RFU Measurements After 3 hours

For Lactose induction of 0 to 10 mM

A comparision of the Standards promoters with pLacI

In comparison with the graphs provided in the Measurement Kit

RFU Measurements After 3.5 hours

For Lactose induction of 0 to 10 mM

A comparision of the Standards promoters with pLacI