Team:Peking/Project/Bioabsorbent/ModuleAssembly

Our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent. To make full use of the space in the bacteria and improve the absorption capacity to a great extent, artificial MBP were constitutively expressed on the surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits. Additionally, MBP will not only expand the accumulation capacity of mercury, but also act as a firewall to against Hg (II) toxity (Fig 1). All the components shown in Fig 1 were assembled together and cloned into pSB3K3 because of the extremely high intensity of T7 promoters.

Fig1. The mercury absorption device we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria. All the components were assembled together and cloned into pSB3K3.

The facilitation module to help our MBPs to meet enough Hg (II) to ensure the binding efficiency was combined with the mercury absorption device. It shares T7 polymerase with the mercury absorption device.

Fig 2: The overall structure of the facilitation module. This module was combined with mercury absorption device mentioned above.

After our bacteria have absorbed enough mercury, a time-delayed aggregation process will be inductively progressed (in response to the presence of mercury at certain concentration) for an easy post treatment such as mercury recycling (Fig 3). This time-delay and amplification device was then cloned into pSB1C3, a high copy plasmid to guarantee the efficiency of aggregation.

Fig 3. The overall structure of the time-delay device. PmerT is a mercury-responsive promoter. In the presence of mercury, the genetic cascade will amplify the signal and express Ag43 with a time delay and high intensity.

These 2 constructs were then transformed into DH5 alpha competent cells. To better demonstrate the function and performance of our bioreporter, we used the characterized indicator for direct visualization of mercury detoxification and bacterial aggregation process, using method described at MBP Expression Page of our wiki.

Fig 4. Direct visualization of mercury detoxification and bacterial aggregation process. Water-soluble metal indicator with colorimetric selectivity for heavy metals was exploited to indicate the mercury concentration in the water. The lower limit of metal concentration for color transition was 0.8×10-5M. (A) The indicator represented rosy color, indicating that high concentration of mercury existed (for more details, see our MBP Expression Page). (B) When applied with our mercury bioabsorbent, after shaking cultured at 37℃ for 10 min, evident color change emerged, indicating that the absorption was under progressing. (C) 30 min later, bacteria aggregated even under slight shaking cultivating at 37℃ in an aslant tube, forming a visible pellet at the bottom. It’s notable that mercury had been decontaminated because the significant color change of the indicator.

In summary, our bioabsorbent worked as expected. It is high-performance for our careful design and eco-friendly because of its inductive aggregation capacity.

Retrieved from "http://2010.igem.org/Team:Peking/Project/Bioabsorbent/ModuleAssembly"

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+Our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent. To make full use of the space in the bacteria and improve the absorption capacity to a great extent, artificial MBP were constitutively expressed on the surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits. Additionally, MBP will not only expand the accumulation capacity of mercury, but also act as a firewall to against Hg (II) toxity (Fig 1). All the components shown in Fig 1 were assembled together and cloned into pSB3K3 because of the extremely high intensity of T7 promoters.
+<html><a href="https://static.igem.org/mediawiki/2010/c/cb/Pkuma1.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/c/cb/Pkuma1.jpg" width=650></a></html><br>
+'''Fig1. The mercury absorption device we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria. All the components were assembled together and cloned into pSB3K3. '''
+The facilitation module to help our MBPs to meet enough Hg (II) to ensure the binding efficiency was combined with the mercury absorption device. It shares T7 polymerase with the mercury absorption device.
+<html><a href="https://static.igem.org/mediawiki/2010/d/d6/Pkuma2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/d/d6/Pkuma2.jpg" width=650></a></html><br>
+'''Fig 2: The overall structure of the facilitation module. This module was combined with mercury absorption device mentioned above. '''
+After our bacteria have absorbed enough mercury, a time-delayed aggregation process will be inductively progressed (in response to the presence of mercury at certain concentration) for an easy post treatment such as mercury recycling (Fig 3). This time-delay and amplification device was then cloned into pSB1C3, a high copy plasmid to guarantee the efficiency of aggregation.
+<html><a href="https://static.igem.org/mediawiki/2010/a/a3/Pkuma3.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/a/a3/Pkuma3.jpg" width=650></a></html><br>
+'''Fig 3. The overall structure of the time-delay device. PmerT is a mercury-responsive promoter. In the presence of mercury, the genetic cascade will amplify the signal and express Ag43 with a time delay and high intensity.'''
+These 2 constructs were then transformed into DH5 alpha competent cells. To better demonstrate the function and performance of our bioreporter, we used the characterized indicator for direct visualization of mercury detoxification and bacterial aggregation process, using method described at MBP Expression Page of our wiki.
+<html><a href="https://static.igem.org/mediawiki/2010/7/71/Pkuma4.png" target="blank"><img src="https://static.igem.org/mediawiki/2010/7/71/Pkuma4.png" width=650></a></html><br>
+'''Fig 4. Direct visualization of mercury detoxification and bacterial aggregation process. Water-soluble metal indicator with colorimetric selectivity for heavy metals was exploited to indicate the mercury concentration in the water. The lower limit of metal concentration for color transition was 0.8×10-5M. (A) The indicator represented rosy color, indicating that high concentration of mercury existed (for more details, see our MBP Expression Page). (B) When applied with our mercury bioabsorbent, after shaking cultured at 37℃ for 10 min, evident color change emerged, indicating that the absorption was under progressing. (C) 30 min later, bacteria aggregated even under slight shaking cultivating at 37℃ in an aslant tube, forming a visible pellet at the bottom. It’s notable that mercury had been decontaminated because the significant color change of the indicator.'''
+In summary, our bioabsorbent worked as expected. It is high-performance for our careful design and eco-friendly because of its inductive aggregation capacity.
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