Team:NCTU Formosa/WetLab/Timer

Result - Timer

Timer Function

A controllable bio-timer which can count variable time duration was constructed in our bacterial referee. The timer function is controlled by the Plac promoter (BBa_R0011), and the concentration of lactose determines timer’s working length. Lactose was used to repress the LacI protein (BBa_C0012) and make the promoter Plac(BBa_R0011) express the down stream genes, cI434(BBa_C0052), tetR(BBa_C0040) and Green Fluorescent Protein (GFP)(BBa_K145015) to implement the timer function. When added lactose is consumed by E. coli, the Red Fluorescent Protein (RFP) start to translate and GFP is degraded. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. The red color in the final step warns us that time’s up and the product lose freshness. A key feature of the bio-timer is that make the longer timing function up to 8-20 hrs.

Component Descriptions

Strand A

1. Constitutive promoter (BBa_J23106) always express the downstream genes, LacI and LuxR (BBa_C0062).
2. LacI inhibits the activity of promoter PLac in the strand B.
3. Complex which combines LuxR with AHL induce the expression of pcIIp22 (BBa_K145150) in strand F.

Strand B

1. In the abscence of lactose, the LacI repressor binds to the promoter Plac(BBa_R0011) and prevents RNA polymerase bound to the promoter (Plac) from transcribing the dawnstream genes. When lactose enters the bacterium, it binds to the LacI repressor and causes a conformational change that inhibits its ability to bind DNA. Consequently, the promoter Plac can transcribe the daenstream genes.
2. The cI repressor (BBa_C0052) inhibits the 434 cI-regulated promoter PcI434 (BBa_R0052) in the strand E.
3. Tetracycline repressor (BBa_C0040) inhibits the tetR-repressible promoter PtetR (BBa_R0040) in the strand C.

Principle and Mechanism

The timer function in our bacterial referee has three stages:

1. Standby phase: bacteria grow in lactose-free medium.
2. Lactose-accession phase: lactose is added in medium.
3. Lactose-consumption phase: bacteria eat all lactose, and the medium does not contain lactose.

1. Standby stage: timer not start yet, and the medium is colorless
   
   
   
   

   For our design, we use lactose as the activator for this bio-timer. When bacteria grow in lactose-free medium, the lacI (BBa_K091121) is produced by constitutive promoter and represses the Plac (BBa_K091110) promoter. In this condition, strand 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 is green

   While E.coli accumulates to the quantity we need (OD=0.1), we add lactose to start our bio-timer. After adding lactose, lactose removes the repression of LacI protein and LacI cannot repress the promoter PLac(BBa_K091110). When lactose is added in the medium, lactose binds with lacI (BBa_K091121) repressor and releases the inhibition of B strand, so the PlacI (BBa_K091110)promoter starts transcripting GFP-LVA (BBa_K145015). Then, the medium are green in this stage.

3. Lactose-consumption phase: the medium is red

   For our bio-timer, the concentration of lactose determines timer’s working length. We need the nature function in E.coli which is the lactose operon and the operons are inherent in E.coli. When lactose exists, the lactose operon is induced for translating the three enzymes, lacZ, lacY and lacA, which are for the degradation of lactose into glucose. To sum up, the bio-timer start to count time after lactose addition and stop working when lactose totally is metabolized because of the lactose operon. When added lactose is consumed by E. coli, the RFP start to translate and GFP is degraded. Therefore, the medium turns from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. The red color in the final step warns us that time’s up.

Present Achivements

The strand A (BBa_K188161) always express the downstream genes, LacI and LuxR (BBa_C0062) and strand B(BBa_K188261) always express the downstream genes, LacI and LuxR (BBa_C0062) were developed and sequenced. However, we do not have more time to keep on further experiments for testing. The experiments we plan to do are as follows:
Since lactose is used as the repressor for LacI protein, 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 A and B strands 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 intensity of green fluorescence after lactose is added. Draw the curve between the time and intensity of green fluorescence.
• Variant duration design
  1. Follow the second experiment, use the different concentrations of lactose to find out the time duration we want.
  2. Contribution of the model between the concentration of lactose and intensity of green fluorescence with time.

Color distinguishing test

Although we can not accomplish the experiments, we still made a 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 culture, the host transformed with BBa_K145279 displalyed obvious green (Fig.1 B); and the host transformed with pSB1A3 displalyed red (Fig.1 D)

Bright green indicates the situation that bio-timer was launched. The red indicates that time’ s up. We assume that when in the transition state from green to red, the RFPs is produced and mix with GFPs, and the medium becomes yellow (Fig.1 C). Therefore,we mixed the tubes of GFPs and RFPs to represent the light-yellow or orange in the transition state. It’s very exciting that we can observe the change of color by our unaimed eyes.

Fig. 1. The predicted spatiotemporal behavior of the bacterial referees. (A) The bacteria grow in colorless lactose-free medium. (B) When lactose is added in medium, lactose binds with lacI repressor and releases the inhibition of GFP. The medium become green in this stage. (C)When added lactose is consumed by E. coli, the RFP start to translate and GFP is degraded. Therefore, the media turn from green color to yellow color (mixed color of green and red light), yellow color to red color step by step. (D) When bacteria eat all lactose, the RFP is expressed by the Plux/cIIp22 promoter, the medium is red in this stage.