In our project, we have characterized two promoters and the cell death device by different methods. This tells us that our parts are functional and we can quantitatively adapt their activity by changing the experimental conditions.

Background Information (link to construct page)
(The basic reason of using this low efficiency constitutive promoter is to enable our bacteria to express a low level of antitoxin so that a certain amount of toxin can be balanced; thus the BMP-2 whose expression is tightly linked with the toxin can be expressed at a certain level as well.)

Objective
Our objective of characterizing this promoter is to determine the relevant activity of it to the standard constitutive promoter activity reference point given by part registry so that we and the subsequent IGEM teams may have an idea how efficient this promoter is.

Method
Instead of measuring the absolute promoter activity, our characterization was generally based on measuring the relevant in vivo activity of this constitutive promoter. By adopting this method, we may be able to eliminate the error caused by different experimental conditions and give a relatively more convincing result.
By linking the promoter with GFP (BBa_E0240), the promoter activity was represented by the GFP synthesis rate which can be easily measured. E.Coli carrying the right construct was then cultured to log phase. During a time slot around the mid-log phase, the GFP intensity and OD595 value were measured to obtain the Relative Promoter Units (RPU).

Procedure

1. Constructing BBa_K733009-pSB3K3 (pTms-BBa_E0240-pSB3K3); Transforming BBa_I20260-pSB3K3 (Standard Constitutive Promoter Activity Reference Point) from 2012 Distribution;
2. Preparing supplemented M9 medium; (link to recipe for supplemented M9 medium)
3. Culturing E.Coli carrying BBa_K733009-pSB3K3 and E.Coli carrying BBa_I20260-pSB3K3 in supplemented M9 medium and measuring the growth curve respectively;
4. Measuring the GFP intensity and ODA595 value every 15 minutes after E.Coli carrying BBa_K733009-pSB3K3 and E.Coli carrying BBa_I20260 are cultured to mid-log phase;
5. Calculating the Relative Promoter Unites (RPU) using the obtained data;
6. Modifying the result.

Result
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Background Information (link to construct page)
(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 the absolute promoter activity and specificity under certain experimental condition.

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. Additionally, the promoter specificity was tested by comparing the induction activity of different chemicals having similar structure to xylose.

Procedure

1. Constructing Pxyl+BBa_E0240-pSB1A2
2. Preparing supplemented M9 medium; (link to Composition for supplemented M9 medium)
3. Culturing E.Coli carrying Pxyl+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 a 3D figure about the GFP intensity unit in respect of xylose concentration and time;
8. Modifying the result;
9. At certain concentration, adding different chemicals having similar structure with xylose to mid log phase bacteria culture;
10. Measuring GFP intensity and OD595 value after 2-hour-induction to evaluate the promoter specificity;
11. Modifying result

Result