Part:BBa_K658005

a bacteria population-control device with RBS0.07 driven by lacl+pL

It is designed to build a programmed bacterial death circuit, which is based on the quorum sensing system of Vibrio fischeri. iGEM-Team XMU-China has designed a series of bacteria population-control devices using RBSs of different strength in the killer protein producer. This device has the ability to maintain cell density of the bacteria population at a relatively low value compared with bacteria without this circuit and extends the steady state. RBS0.07 (BBa_B0031) was used to regulate the expression of killer protein ccdB.

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Figure 1. Animation of proposed reaction mechanism of iccdB0.07

Description

This device is made up of three subparts: a LuxI producer, a LuxR producer and a killer protein producer. The LuxI protein synthesizes a small, diffusible acyl-homoserinelactone (AHL) signaling molecule. The AHL accumulates as the cell density increases. At sufficiently high concentrations, it binds the LuxR, which induces the expression of the killer gene ccdB under the control of a promoter lux pR. Sufficiently high levels of CcdB which is a bacterial toxin that targets DNA gyrase cause cell death. Low cell density doesn’t have the ability to produce sufficient LuxR/AHL complex to activate the promoter lux pR. The programmed death circuit ends and the cell density increases. When the cells reach a certain concentration, the death circuit is restarted. Back and forth, the programmed death is achieved in the dynamic process of growth and death. In that way, the bacteria population is programmed to maintain one certain cell density.

Performance

In order to test the performance of the bacteria population-control device iccdB0.07, this device was first cloned into plasmid pSB1A2, followed by transformation into E.coli strain BL21.

The cell growth curves in Figure 2 showed the bacteria population-control device successfully maintained the cell density at a lower value at the steady state compared with BL21’s cells without this circuit. Besides, circuit-regulated cell growth (black dot) has a relatively longer steady state than cells without this circuit (red dot).

For iccdB0.07 regulated cell growth, the viable cell density first start to decline around 8h and then reached a steady state after two minor oscillations. Compared with bacterium without this circuit, the iccdB0.07 regulated bacterium has a lower media-consuming rate due to its lower cell density.

The iccdB0.07 regulated cell growth might be explained by “ON-OFF” mechanism based on the quorum sensing system.

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Figure 2 Experimentally measured growth curves of BL21’ cells without iccdB0.07 (red dot) and with iccdB0.07 ON (black dot).

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Figure 3 Experimentally measured densities of BL21’s cells without population-control circuit and BL21’s cells with four population-control circuits respectively at steady state.

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Figure 4 Average of experimentally measured cell densities of BL21’s cells with four population-control circuit at steady state.

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Table 1 Data of the steady-state cell density.

iGEM Team XMU-China has also tested the performance of three other bacteria population-control device iccdB1.0 (BBa_K658001), iccdB0.6 (BBa_K658003) and iccdB0.3 (BBa_K658004). Figure 3, figure 4 and table 1 illustrate that by using RBS of different strength in the population-control device, we were able to control the steady-state cell density of a bacteria population at different levels. And a population-control device with RBS of high strength results in a low steady-state cell density. It might be explained by the mechanism that for circuit-regulated growth, the cell death rate is regarded proportional to the intracellular concentration of the killer protein. The expression of the killer gene is regulated by the strength of its upstream RBS. Therefore, a RBS with higher strength promises more killer protein in vivo, which leads to a higher death rate of the bacteria population.

Method

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 718
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2346
  Illegal BsaI.rc site found at 1874