Difference between revisions of "Part:BBa K733018"

Revision as of 06:31, 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 of using xylose inducible promoter is to make the expression of toxin and BMP-2 controllable. Xylose is not toxic and normally is not present in human colon. This provides us an easy way to induce BMP-2 expression without disrupting normal human body function.

Objective

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.

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 should be observed.

Method

The absolute promoter activity was measured in respect to induction time and 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.

Characterization Procedure

1. Constructing xylR-PxylA-BBa_E0240-pSB1A2

2. Preparing supplemented M9 medium (see below);

3. CulturingE.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 same bacteria in supplemented M9 medium to log phase;

5. Adding xylose at different concentration to different sets of culture medium;

6. Measuring the GFP intensity and OD595 value across time for every sets of culture medium that are of different xylose concentration;

7. Plotting afigure about the GFP intensity unit in respect of xylose concentration and time;

8. Compiling the result.

Data Processing

1. After E.coli carrying the right was growing into mid-log phase, GFP intensity and ODA595 were measured every 30 minutes (up to 120min);

2. For GFP intensity, curve reflecting GFP expression change was plotted; for ODA595, average value was taken;

3.GFP synthesis rate was then represented by the slope of the curve reflecting GFP expression change;

4. Absolute promoter activity for the promoter under different inducer concentrations were calculated by divide the corresponding GFP synthesis rate by the average ODA595 value;

5. Absolute promoter activity was then modified by taking the average value of all sets of data obtained.

Result

1. 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.

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

3. 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%

4. 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)

Discussion

1. 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.

2. 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.

3. 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.

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