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 Regulation and Control Module)
(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 for characterizing this promoter is to test whether pTms works in E.coli DH10B strain and determine the relative promoter units (RPU) 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.

Intended Result
1. pTms should work in E.coli. This is supported by previous research (Moran et al., 1982).
2. The activity of pTms should be relatively low.

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).

Characterization 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 (see below);
3. Culturing E.coli DH10B strain 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. Compiling the result.

Data Processing

1. After E.coli carrying the right construct was grown into mid-log phase, GFP intensity and ODA595 were measured every 15 minutes (up to 60min);
2. For GFP intensity, curve reflecting GFP expression change was plotted; for ODA595, average values was taken;
3. GFP synthesis rate was then represented by the slope of the curve reflecting GFP expression change;
4. Absolute promoter activity of pTms and I20260 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;
6. Finally, R.P.U was calculated by dividing pTms absolute promoter activity by I20260 absolute promoter activity.

Result
1. Suggested by the GFP expression curve we plotted, pTms functions in E.coli DH10B strain.
Picture
* GFP expression curve for one set of data
2. The overall RPU was calculated as 0.046497. It has been shown that pTms has a very low promoter efficiency in E.coli DH10B stain.
Picture.

Discussion
Compared with I20260, it seems that bacteria carrying pTms had a rather low GFP expression. This may cause some difficulty upon deciding whether pTms functions in E.coli or not. However, since viewed from the curve, the GFP expression for pTms increased gradually in respect to time. These all give us good reason to say that pTms functions in E.coli DH10B strain, although with a low efficiency. Another reason for its low efficiency could be that pTms was originally got from B.subtilis and is only suggested to be functional in E.coli. While we haven’t got time to characterize the promoter in B.subtilis, we still hope that in the future we can actually achieve that.

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

Background Information (link to construct page)

Objective
Upon characterization, we want to know what concentration of xylose can induce the death of cells carrying our ydcDE device. It does not necessarily need to be a minimal thresh hold, as long as a concentration for killing the bacteria is defined, we can come to the conclusion that xylose concentration which is above the point can kill the cell or at least inhibitory the cell growth.

Method
We assembled ydcD and ydcE into the same plasmid as our artificial operon. Bacteria carrying the right construct were then cultured in supplemented M9 medium of serial xylose concentration. By comparing the turbidity of experimental group with the negative control, we got a concentration of xylose above which cells would dye.

Procedure

1. Constructing ^^^^;
2. Culturing bacteria carrying the right construct in supplemented M9 medium of serial xylose concentration;
3. Checking the turbidity of cell culture after ^^ hours;
4. Doing several more round of experiment and modifying the result.

Result