Team:NCTU Formosa/WetLab/Timer

Result - Timer

In our project, at first, we need a timer which can count different times we need. Base on this purpose, we decided to use lactose to repress the LacI(C0012) and make pLacI(R0011) express the down stream genes, cI434(C0052), tetR(C0040) and GFP(K145015) to lauch the timer function and control the duration of counting time. In addition, we plan to find out the different time we could control by using variant concentrations of lactose.

Component Descriptions

Device A

1. Constitutive promoter(J23106) always express the downstream genes, LacI and LuxR(C0062).
2. LacI inhibits the pLacI expression in the Device B(K188261).
3. LuxR combines to AHL to form the complex which induce the expression of pcIIp22(K145150) in Device F.

Device B

1. acI-regulated promoter (R0011) is to be inactive in the presence of LacI, and when lactose added binds LacI, the promoter will work.
2. The cI repressor (C0052) inhibits the 434 cI-regulated promoter(R0052) in the Device E.
3. Tetracycline repressor (C0040) inhibits the tetR-repressible promoter(R0040) in the Device C.
4. Green fluorescent protein (K145015) expression makes us know that the Timer starts to count.

Timer Mechanisms

There are two phases during our timer is working:

1. Standby phase: the medium are colorless
   

   For our design, we use lactose as the repressor for this chemical timer. Before counting the time, lactose does not be added to the medium, we can know that LacI in Device A is expressed for repressing the promoter LacI in the Device B. In this condition, Device B does not work and also GFP‧LVA is not expressed. During this time, E.coli does nothing but grow and cells in the medium are colorless.


2. Lactose-accession phase: the medium are green
   

   However, while E.coli accumulates to the quantity we need (OD=0.1), we add lactose to start our chemical timer. As a repressor, lactose removes the repression of LacI to promoter LacI. After adding lactose, LacI cannot repress the promoter LacI when the lactose exists. Promoter Lac switches on the downstream genes expression in this condtion. For our chemical timer, we need the nature function in E.coli which is the lactose operon and the operon is inherent in E.coli. When lactose exists, lactose operon will be induced for translating the three enzymes, lacZ, lacY and lacA, which are for the degradation of lactose into glucose. To sum up, the timer will start to count time after lactose addition and stop working when lactose totally is metabolized because of the lactose operon.








Present Achivements

Until now, we have finished the construction of our Device A(K188161) and Device B and delivered them to be sequenced. Unfortunately, we do not have more time to keep on further experiments for testing. The experiments we plan to do are followings.
     Because we use lactose as the repressor for LacI, the carbon source in the broth can not be glucose or any carbon source will be the priority of use to lactose. Therefore, we choose the glycerol as our carbon source in M9 medium for the experiment.

• Growth curve determination
  1. The first and second devices are transformed to the host cells, and the cells are cultured in the M9 medium with glycerol as the carbon source to determine growth curve.
  2. Contribution of the curve between time and O.D. (optical density).
• Time-delay examination
  1. Base on the above growth curve, add the lactose to final concentration is 1mM (compared with IPTG induction concentration) when O.D. of culture arrive to 0.1.
  2. Detect the green-fluorescent after lactose are added. Draw the curve between the time and intensity of green-fluorescent.
• Variant duration design
  1. Follow the second experiment, use the different concentrations of lactose to find out the time we want.
  2. Contribution of the model between the concentration of lactose and intensity of green-fluorescent with time.






Timer Function Simulation

Based on the mechanism we designed for the timer, we made a simulation about the counting-time function with different concentrations of lactose. This simulation is constructed by four of six devices in our system. They are A,B,C and F (see the figure below).



According to the diagrams, the simulations almost tally with our expectations. In figure A, the expressing intensity of GFP and the duration of timer functioning decrease with the concentration of lactose added decreasing. Moreover, the counting-time can be controlled from 20 hours to 8 hours. In the other figure B, the intensity of GFP is the same for different concentrations of AHL, but the expression of mRFP to the AHL-LuxR complex is quicker with higher concentration of AHL.



Figure A. The expressing intensity of GFP and the duration before mRFP expression decrease with lactose added decreases. The unit of [lactose] is not specific, and it’s an assumed amount.



Figure B. The expression of mRFP to the AHL-LuxR complex is quicker with higher concentration of AHL. αout=outside [AHL], AHL is supposed to be the external harm bacteria.







Color Distinguishing Test

Although we can not accomplish the experiments, we still made a little color test to explain the possible result we expect. We cultured two tubes with host cells over night,and the host in one tube was transformed with BBa_K145279 and the other with pSB1A3. Over one night, the color of tubes is very apparent and we can easily tell the colors. The images below are the tubes we cultured.



(Left) The green tube with the host transformed with BBa_K145279. (Middle) The red tube with the host transformed with pSB1A3. (Right) From the left to the right: control tube(without inducer, K145279 tube, mixed tube, pSB1A3 tube.

The green tube presented the situation that our timer was launched. The red one presented that the time was up. Then we assumed that when time is up, the red flurorenscent proteins would be produced and then mix with green flurorensent proteins.As a result, the tube would be yellow.

Therefore,we mixed the two tubes by drops into a new tube, and we can see the color of the mixed tube is light-yellow or orange. It’s very exciting that we can just observe the change of color by our unaimed eyes.