Team:XMU-China/timedelay

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       <p align="center"><img src="https://static.igem.org/mediawiki/2012/d/d6/TimedelayFig5.png" alt="" width="452" height="322" /><br />
       <p align="center"><img src="https://static.igem.org/mediawiki/2012/d/d6/TimedelayFig5.png" alt="" width="452" height="322" /><br />
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        Figure 5. Fluorescence curves of strain TD1.0 induced by 0.1 mM arabinose  and strain TD1.0 without arabinose.<br />
 
         <img src="https://static.igem.org/mediawiki/2012/e/ea/TimedelayFig_6.png" alt="" width="457" height="300" /><br />
         <img src="https://static.igem.org/mediawiki/2012/e/ea/TimedelayFig_6.png" alt="" width="457" height="300" /><br />
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        Figure  6. Fluorescence curves of strain TD0.01 induced by 0.1 mM arabinose and strain TD0.01 without  arabinose.</p>
 
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       <p align="center"><img src="https://static.igem.org/mediawiki/2012/c/c5/TimedelayFig_7.png" alt="" width="517" height="367" /><br />
       <p align="center"><img src="https://static.igem.org/mediawiki/2012/c/c5/TimedelayFig_7.png" alt="" width="517" height="367" /><br />
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         Figure 7. Fluorescence curves of strain TD0.6 induced by 0.1 mM arabinose and strain TD0.6 without arabinose</p>
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         Figure 5.Fluorescence curves of strain TD1.0， TD0.6 and TD0.01 induced with 0.1 mM arabinose and without arabinose.</p>
       <p align="center"><img src="https://static.igem.org/mediawiki/2012/3/3c/TimedelayFig_8.png" alt="" width="449" height="334" /><br />
       <p align="center"><img src="https://static.igem.org/mediawiki/2012/3/3c/TimedelayFig_8.png" alt="" width="449" height="334" /><br />
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         Figure  8. Fluorescence curves of the induced strain TD0.01, TD0.6, TD1.0 and <em>E. coli</em> BL21(DE3)<br />
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         Figure  6. Fluorescence curves of the induced strain TD0.01, TD0.6, TD1.0 and <em>E. coli</em> BL21(DE3)<br />
       <img src="https://static.igem.org/mediawiki/2012/0/00/TimedelayFig_9.png" alt="" width="480" height="371" /><br />
       <img src="https://static.igem.org/mediawiki/2012/0/00/TimedelayFig_9.png" alt="" width="480" height="371" /><br />
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         Figure  9. Experimentally measured the average time of the three cultures&rsquo; fluorescence  up to 5000 <br>
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         Figure  7. Experimentally measured the average time of the three cultures&rsquo; fluorescence  up to 5000 <br>
         after induction, it shows that the stronger RBS circuit contains,  the less time it takes.</p>
         after induction, it shows that the stronger RBS circuit contains,  the less time it takes.</p>
       <p>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 RBS<sub>0.01</sub>, 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  strain TD1.0 was the highest, strain TD0.6 was middle and strain 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.</p>
       <p>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 RBS<sub>0.01</sub>, 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  strain TD1.0 was the highest, strain TD0.6 was middle and strain 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.</p>

Revision as of 12:33, 26 September 2012

XMU-CSS

XMU

timeindex

Contents[hide][show]
  • 1. Overview
    		•
  • 2. Review of last year
    		•
  • 3. Constructions this year
  • 4. Results and discussion
    		•
  • 5. Reference
    		•
    timedelay

    Time Delay

    1. Overview

    For the lengths of our genetic circuits are not the same, based on quorum sensing system we had studied last year, we considered that the time of genetic expressions of each circuit is different because of the different lengths and strength of ribosome binding sites. So we constructed circuits with three grades strength of ribosome binding sites, including 0.01, 0.6 and 1.0. After induced by arabinose, the duration of response time for GFP expression increased as the strength of RBS declined, bringing about a time-course display.

     

    2. Review of last year

    Last year, we had developed a series of devices which program a bacteria population to maintain at different cell densities. [1]We had designed and characterized the genetic circuit to establish a bacterial ‘population-control’ device in E. coli based on the well-known quorum-sensing system from Vibrio fischeri, which autonomously regulates the density of an E. coli population. The cell density, however, was influenced by the expression levels of a killer gene (ccdB) in our device. As such, we had successfully controlled the expression levels of CcdB by using RBSes of different strength and mutated luxR promoters (lux pR). This circuit incorporated a mechanism for programmed death in respond to changes in the environment.


    Figure 1. The mechanism graph of iccdB last year

     

     

     

    3. Constructions this year


    Figure 2. The construction devices of time delay system

    Based on our last year’s project, for convenience of testing the results of time delay device, we chose GFP instead of ccdB as our reporter. On the other hand, because of the basal expression of Plac is very strong, we decided to use arabinose to induce PBAD promoter to activate our device. Meanwhile, to accomplish the aim of time delay, which means we should make the GFP produce in different period of time, we decided to change the RBS’s strength of luxI expression system. RBSes of different strengths will cause the speed of quorum sensing effect, which will lead to the delay of GFP production time.

    Figure 3. The mechanism graph of time delay system

    4. Results and discussion

    4.1 Optimization of inducer concentration

    Figure 4.Effects of arabinose concentration on the fluorescence of strain TD1.0.

    We can find that the curve with 0.1 mM arabinose has a higher fluorescence. So we chose the 0.1 mM arabinose for the following fluorescence tests.

    4.2 Fluorescent test of the time delay devices






    Figure 5.Fluorescence curves of strain TD1.0， TD0.6 and TD0.01 induced with 0.1 mM arabinose and without arabinose.


    Figure 6. Fluorescence curves of the induced strain TD0.01, TD0.6, TD1.0 and E. coli BL21(DE3)

    Figure 7. Experimentally measured the average time of the three cultures’ fluorescence up to 5000
    after induction, it shows that the stronger RBS circuit contains, the less time it takes.

    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 strain TD1.0 was the highest, strain TD0.6 was middle and strain 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.

    5. Reference

    https://2011.igem.org/Team:XMU-China

     

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