Difference between revisions of "Part:BBa K733018"

Revision as of 23:11, 26 September 2012

xylR+PxylA+RBS+GFP+Double Terminator

This construct is built to measure the relative efficiency of xylose inducible promoter in E. coli. We intend to use GFP as an indicator to characterize this xylose inducible promoter derived from Bacillus megaterium (BBa_K733002). In our characterization, we will investigate the influence of concentration of xylose on xylose inducible promoter and use GFP intensity changes over certain period of time to represent the regulatory effect of this promoter in different xylose inducing conditions.

Characterization

Background Information [http://2012.igem.org/Team:HKUST-Hong_Kong/Module/Regulation_and_control Link to our Regulation and Control Module]

The reason for using the xylose inducible promoter is to enable control on the expression of toxin and BMP2. Xylose is not toxic and normally not present in the human colon. This provides us an easy way to induce BMP2 expression without disrupting normal human body function.

Objective On characterization, we want to test whether the promoter works in E. coli DH10B strain and if it works, what is the absolute promoter activity under varied experimental condition (i.e. xylose concentration).

Intended Result

1. Xylose inducible promoter is functional in E.coli.

2. After the inducer concentration has reached certain level, a relatively stationary GFP expression level (expression upper-limit should be observed.

Method

The absolute promoter activity was measured with respect to xylose concentration.

The same reporter gene (BBa_E0240) was used to indicate promoter activity. E. coli carrying the right construct was cultured to log phase. Following the addition of xylose at various predetermined concentrations, at a time point around the mid-log phase, the GFP intensity and OD595 were measured for every 30 mins (up to 120 mins). Independent curves indicating the GFP intensity units (of various xylose concentrations) with respect to time were then plotted, following which the respective absolute promoter activities were calculated.

Characterization Procedure

1. Constructing xylR-PxylA-BBa_E0240-pSB1A2; 2. Preparing supplemented M9 medium (see below); 3. Culturing E. coli carrying xylR-PxylA-BBa_E0240-pSB1A2 and E. coli without constructs in supplemented M9 medium and measuring the growth curve respectively; 4. Culturing the above mentioned bacteria in supplemented M9 medium to log phase; 5. Adding xylose at different concentrations to different sets of bacterial culture; 6. Measuring the GFP intensity and OD595 values across time for every set of bacterial culture containing different xylose concentrations; 7. Plotting independent curves showing the GFP intensity units of various xylose concentrations with respect to time; 8. Plotting a graph to demonstrate the absolute promoter activity under different inducer concentrations; 9. Compiling the results.

Data Processing

1. After E. coli carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120 mins);

2. For GFP intensity, curve reflecting GFP expression change was plotted; for OD595, average value was taken;

3. GFP synthesis rate was then obtained by calculating the slope of linear regression line of the above mentioned curve;

4. Absolute promoter activity for the promoter under different inducer concentrations were calculated by dividing the corresponding GFP synthesis rate over the average OD595 value;

5. Averaged absolute promoter activity was then obtained by averaging the respective sets of absolute promoter activity values.

Result

1. Shown in the figure below, with the addition of xylose, GFP expression increased. This tells us that the xylose inducible promoter is functional in E. coli DH10B strain.

2. When no xylose was added, a limited amount of GFP was expressed. This suggests that the xylose inducible promoter is to some extent leaky.

3. A relatively stationary GFP expression level was observed at xylose concentrations of 1% to 5%. Despite some other variables (see discussion for more details), the data suggests that the minimum inducer concentration for triggering a full induction should lie somewhere between 0% and 1%.

4. For 10% inducer concentration, the GFP expression was relatively lower. There could be several reasons for this occurence, such as suboptimal growth conditions due to high osmotic pressure. (See discussion for more details.)

Discussion

1. It is quite obvious that addition of xylose induces GFP expression in this construct. However, a slight issue remains: even when no xylose was added, a minute but detectable amount of GFP was still expressed. This shows that the xylose inducible promoter is leaky. It should be noteworthy that this version of the xylose inducible promoter has undergone mutagenesis on 3 different sites on the repressive gene for standardization purposes. Even so, the mutagenesis is done while preserving an identical codon translation. As such, similar to the Ptms promoter, activity of the xylR-PxylA promoter might be different in E. coli from that of B. subtilis due to expression in a heterologous system. Further characterization on this promoter in B. subtilis would be our future aim as time was limited.

2. Another interesting fact to note is that the E. coli strain used is one in which it's xylose metabolic operon remains active. As such, one might assume that the observed GFP expression upper-limit ("stationary expression level"),particularly at 1% or 2% is not the "true" upper-limit, since there should be underlying metabolism of xylose. In order to eliminate this possible error, higher concentrations of xylose was used and the promoter activity does not appear to vary greatly, suggesting that the stationary expression level reflects the maximum promoter activity. While this problem is solved, due to the relatively large concentration difference of inducer, it appears that the maximum promoter activity was achieved at 1% xylose, implying that we are unable to determine the exact minimum inducer concentration requirement for maximum activity. Based on the current result, it is safe for us to make a conjecture that the minimum concentration required lies between 0% and 1% xylose.

3. For the decreased GFP expression at 10% xylose concentration, one possible reason is that the high osmotic pressure caused by the xylose in the medium may inhibit the growth and metabolism of bacteria, thus reducing the bacterial population and/or its GFP expression. Another possible, but unlikely reason could be that the over-expression of induced GFP expression may disturb the normal bacterial function, leading to a low overall GFP expression.

Supplemented M9 Medium Composition

1. 5X M9 Salt Composition (1L)

(1) 64g Na₂HPO₄﹒

(2) 15g KH₂PO₄

(3) 2.5g NaCl

(4) 5.0g NH₄Cl

2. Minimal 1X M9 medium (1L)

(1) 200ml of 5X M9 Salt

(2) 2ml of 1M MgSO₄

(3) 100μl of 1M CaCl₂

(4) 5ml of 40% glycerol

3. Supplement (for the final medium)

(1) 1mM thiamine hydrochloride

(2) 0.2% casamino acids

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal XhoI site found at 847
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 2058

@@ Line 11: / Line 11: @@
 '''Background Information''' [http://2012.igem.org/Team:HKUST-Hong_Kong/Module/Regulation_and_control Link to our Regulation and Control Module]
+The reason for using the xylose inducible promoter is to enable control on the expression of toxin and BMP2. Xylose is not toxic and normally not present in the human colon. This provides us an easy way to induce BMP2 expression without disrupting normal human body function.
-The reason of using xylose inducible promoter is to make the expression of toxin and ''Bmp2'' controllable. Xylose is not toxic and normally is not present in human colon. This provides us an easy way to induce ''Bmp2'' expression without disrupting normal human body function.
 '''Objective'''
+On characterization, we want to test whether the promoter works in E. coli DH10B strain and if it works, what is the absolute promoter activity under varied experimental condition (i.e. xylose concentration).
-Upon characterizing xylose inducible promoter, we want to test whether xylose inducible promoter works in ''E.coli'' DH10B strain and if it works what is the absolute promoter activity under certain experimental condition.
@@ Line 24: / Line 22: @@
 . Xylose inducible promoter is functional in ''E.coli''.
-. After the inducer concentration has reached certain level, a relatively stationary GFP expression level should be observed.
+. After the inducer concentration has reached certain level, a relatively stationary GFP expression level (expression upper-limit should be observed.
 '''Method'''
-The absolute promoter activity was measured in respect to induction time and xylose concentration.
+The absolute promoter activity was measured with respect to xylose concentration.
-Here the same reporter gene (BBa_E0240) was used to indicate promoter activity.'' E.coli ''carrying the right construct was cultured to log phase. Following the addition of xylose at serial concentration, during a time slot around the mid log point, the GFP intensity and ODA595 were measured for every 30 min. A curve indicating the GFP intensity unit as a respect of time and xylose concentration was plotted.
+The same reporter gene (BBa_E0240) was used to indicate promoter activity. E. coli carrying the right construct was cultured to log phase. Following the addition of xylose at various predetermined concentrations, at a time point around the mid-log phase, the GFP intensity and OD595 were measured for every 30 mins (up to 120 mins). Independent curves indicating the GFP intensity units (of various xylose concentrations) with respect to time were then plotted, following which the respective absolute promoter activities were calculated.
 '''Characterization Procedure'''
-. Constructing ''xylR''-''PxylA''-BBa_E0240-pSB1A2
+. Constructing xylR-PxylA-BBa_E0240-pSB1A2;
 . Preparing supplemented M9 medium (see below);
+. Culturing E. coli carrying xylR-PxylA-BBa_E0240-pSB1A2 and E. coli without constructs in supplemented M9 medium and measuring the growth curve respectively;
-. Culturing''E.coli'' carrying ''xylR''-''PxylA''-BBa_E0240-pSB1A2 and'' E.coli ''without constructs in supplemented M9 medium and measuring the growth curve respectively;
+. Culturing the above mentioned bacteria in supplemented M9 medium to log phase;
+. Adding xylose at different concentrations to different sets of bacterial culture;
-. Culturing the same bacteria in supplemented M9 medium to log phase;
+. Measuring the GFP intensity and OD595 values across time for every set of bacterial culture containing different xylose concentrations;
+. Plotting independent curves showing the GFP intensity units of various xylose concentrations with respect to time;
-. Adding xylose at different concentration to different sets of culture medium;
+. Plotting a graph to demonstrate the absolute promoter activity under different inducer concentrations;
+. Compiling the results.
-. Measuring the GFP intensity and OD595 value across time for every sets of culture medium that are of different xylose concentration;
-. Plotting afigure about the GFP intensity unit in respect of xylose concentration and time;
-. Compiling the result.
 '''Data Processing'''
-. After'' E.coli'' carrying the right was growing into mid-log phase, GFP intensity and ODA595 were measured every 30 minutes (up to 120min);
+. After E. coli carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120 mins);
-. For GFP intensity, curve reflecting GFP expression change was plotted; for ODA595, average value was taken;
+. For GFP intensity, curve reflecting GFP expression change was plotted; for OD595, average value was taken;
-.GFP synthesis rate was then represented by the slope of the curve reflecting GFP expression change;
+. GFP synthesis rate was then obtained by calculating the slope of linear regression line of the above mentioned curve;
-. Absolute promoter activity for the promoter under different inducer concentrations were calculated by divide the corresponding GFP synthesis rate by the average ODA595 value;
+. Absolute promoter activity for the promoter under different inducer concentrations were calculated by dividing the corresponding GFP synthesis rate over the average OD595 value;
-. Absolute promoter activity was then modified by taking the average value of all sets of data obtained.
+. Averaged absolute promoter activity was then obtained by averaging the respective sets of absolute promoter activity values.
 '''Result'''
-. Shown by our figure below, under the addition of xylose, GFP expression increased. This tells us that xylose inducible promoter is functional in'' E.coli DH10B ''strain.
+. Shown in the figure below, with the addition of xylose, GFP expression increased. This tells us that the xylose inducible promoter is functional in E. coli DH10B strain.
-. When no xylose was added, a little amount of GFP was expressed. This suggests that xylose inducible promoter is to some extent leaky.
+. When no xylose was added, a limited amount of GFP was expressed. This suggests that the xylose inducible promoter is to some extent leaky.
-. A relatively stationary GFP expression was observed after xylose concentration increased to 1% up to 5%. Despite some other variables (see discussion for more detail), we would say that the minimum inducer concentration for triggering full induction should lie in somewhere between 0 and 1%
+. A relatively stationary GFP expression level was observed at xylose concentrations of 1% to 5%. Despite some other variables (see discussion for more details), the data suggests that the minimum inducer concentration for triggering a full induction should lie somewhere between 0% and 1%.
+. For 10% inducer concentration, the GFP expression was relatively lower. There could be several reasons for this occurence, such as suboptimal growth conditions due to high osmotic pressure. (See discussion for more details.)
-. For 10% inducer concentration, the GFP expression was relatively lower. There could be several reasons for that, such as xylose metabolism by bacteria. (see discussion for more detail)
 [[Image:PXylGFP.jpg]]
@@ Line 81: / Line 74: @@
 '''Discussion'''
-. It is quite obvious that addition of xylose apparently induces the GFP expression. However, problem lies in that even when no xylose was added, a detectable amount of GFP was still expressed. This means that xylose inducible promoter was leaky. Reason for this could be that three mutagenesis had been done to the repressive gene of this promoter. Although we have adopted the most frequently used codon in ''B.subtilis'' for the mutagenesis, this may not work as our expectation in ''E.coli''.
+. It is quite obvious that addition of xylose induces GFP expression in this construct. However, a slight issue remains: even when no xylose was added, a minute but detectable amount of GFP was still expressed. This shows that the xylose inducible promoter is leaky. It should be noteworthy that this version of the xylose inducible promoter has undergone mutagenesis on 3 different sites on the repressive gene for standardization purposes. Even so, the mutagenesis is done while preserving an identical codon translation. As such, similar to the Ptms promoter, activity of the xylR-PxylA promoter might be different in E. coli from that of B. subtilis due to expression in a heterologous system. Further characterization on this promoter in B. subtilis would be our future aim as time was limited.
-. For the observation of full induction. Our biggest problem is that the ''E.coli'' strain we used contains the xylose metabolic operon, which means xylose might be metabolized by the bacteria. To eliminate error caused by this factor, we chose to use relatively higher concentration for experiment. This further caused another problem on determining the minimum xylose concentration for full GFP induction as when xylose concentration increased to 1%, the observed GFP expression level already entered a relatively stationary phase. Therefore, based on this result, we would say that due to bacterial metabolism of xylose, we are not sure whether the real GFP maximum level is higher than our current observation. However, since for a xylose concentration above 1%, a relative stationary level of GFP was observed, we would say that the minimum xylose concentration to trigger the full induction lies below 1%. We hope that in the future we can confirm the exact concentration.
+. Another interesting fact to note is that the E. coli strain used is one in which it's xylose metabolic operon remains active. As such, one might assume that the observed GFP expression upper-limit ("stationary expression level"),particularly at 1% or 2% is not the "true" upper-limit, since there should be underlying metabolism of xylose. In order to eliminate this possible error, higher concentrations of xylose was used and the promoter activity does not appear to vary greatly, suggesting that the stationary expression level reflects the maximum promoter activity. While this problem is solved, due to the relatively large concentration difference of inducer, it appears that the maximum promoter activity was achieved at 1% xylose, implying that we are unable to determine the exact minimum inducer concentration requirement for maximum activity. Based on the current result, it is safe for us to make a conjecture that the minimum concentration required lies between 0% and 1% xylose.
-. For the GFP expression decrease at 10% xylose concentration, one possible reason is that the high osmotic pressure caused by the medium may inhibit the growth and metabolism of bacteria, thus reducing the GFP expression. Another possible reason could be that the over expression of induced GFP expression may disturb the normal bacteria function, leading to a low overall GFP expression.
+. For the decreased GFP expression at 10% xylose concentration, one possible reason is that the high osmotic pressure caused by the xylose in the medium may inhibit the growth and metabolism of bacteria, thus reducing the bacterial population and/or its GFP expression. Another possible, but unlikely reason could be that the over-expression of induced GFP expression may disturb the normal bacterial function, leading to a low overall GFP expression.