Part:BBa_K750012

TIME DELAY0.01:LuxI(RBS0.01)->LuxR->LuxPR->GFP

Description

This part contains BBa_K750004 and BBa_K750007. Arabinose will activate the promoter pBAD, which will cause production of protein LuxI and LuxR. The protein LuxI will synthesize a small, diffusible signaling molecule acyl-homoserinelactone(AHL). The AHL accumulates as the cell density increases. At sufficiently high concentrations, it binds the LuxR, which induces the expression of our reporter GFP under the control of a promoter lux pR. Expression of LuxI will be affected when strength of RBS before luxI gene is changed. When expression of luxI is weak, speed of AHL production will go down, that means it costs longer time for AHL-LuxR to arrive the activation thrshold of the promoter lux pR. In the time delay part of our project E.Lumoli, we designed 4 circuit with RBSes of different strength in the LuxI producer to construct a time delay device. Other time delay parts: TD1.0(BBa_K750009), TD0.6(BBa_K750010), TD0.3(BBa_K750011).

Performance

We had took a pre-experiment before we did the fluorescence test. We useed TD1.0 as the experimental subject, and inducted it by 0.1mM, 1.0mM, 10mM arabinose.

Figure 1. The optimization of the arabinose induced concentration added into the TD1.0 including 0, 0.1, 1.0, 10 mM arabinose.

From this figure, we ensured that 0.1mM arabinose induct the circuit best. So we finally chose 0.1mM arabinose for fluorescence test later.
Then we did the fluorescence test of TD1.0.

Figure 2. Fluorescence curves of TD0.01 induced by 0.1mM arabinose and TD1.0 without arabinose.

Prepared with its control, fluorescence value of TD1.0 arrived at a much higher degree. That means the GFP has been produced well, the whole circuit are working well.
At the same time, we did fuorescence test of another two circuit we had finished construction:TD1.0(BBa_K750009), TD0.01(BBa_K750012).

Figure 3. Fluorescence curves of TD1.0 induced by 0.1mM arabinose and TD0.01 without arabinose.

Figure 4. Fluorescence curves of TD0.6 induced by 0.1mM arabinose and TD1.0 without arabinose

In order to prove that there is a time delay phenomenon when these 3 circuit working, we put curves of them into one figure:

Figure 5. Fluorescence curves of the induced TD0.01, TD0.6, TD1.0 and the control BL21

We chose the value of fluorescence 5000 to compare. Here is a column chart to display how long these 3 circuit take to arrive the value 5000:

Figure 6. Experiemently measured the average time of the three cultures’ fluorescence up to 5000 after induction.

And we can find that there surely a time delay phenomenon when these 3 circuit working together. They are working as we designed.
Compared with the groups absence of arabinose, the fluorescence of induction groups were much higher. On the other hand, we could find even the circuit with RBS0.01, the most insensitive one, also expressed green fluorescence protein without arabinose, which means basal expressing phenomenon. According to figure 5, when the three cultures induced by the same concentration of arabinose, the fluorescence of TD1.0 was the highest, TD0.6 was middle and TD0.01 was the lowest. Finally, as shown in figure 8, the fluorescence of the circuits with different RBSes also reached a certain level in different time. It fit the goal of time delay.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 125
  Illegal NheI site found at 1068
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 792
  Illegal BamHI site found at 65
  Illegal BamHI site found at 1008
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2023
  Illegal BsaI.rc site found at 2750