|Projects||Design: Wet Lab||Design: Printer||Design: Software||Testing||Construction: The Wetlab||Protocols||Final Result of E.co Lisa|
Welcome to our Teams wetlab section. Our wetlab entry consists of several different components, which are described in this section. Just click on the component you want to learn more about
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The success of our project is dependent on us having very precise control over the expression of our reporter gene. In order to draw a legible picture our system needs to be able to induce very high expression of agarase under the desired conditions and then almost completely stop agarase expression when the activating conditions are removed. This is because an over expression of agarase might just degrade the entire plate leaving nothing visible as a picture. Initially one of our team members, Dave Curran, designed a very slick and complex system to regulate expression. However, as we were running out of time we were forced to rely on a simpler version. The simplified version works as follows.
In the absence of 660nm light and AHL, both promoters in the circuit are repressed. If the bacteria were then to be exposed to light, the protein LuxR would be produced, but would not activate the promoter lux pR. When AHL is added to the cells that are still in the dark, the lux pR promoter would still not be activated, as the protein LuxR would not be present. But if AHL is added to the cells, and they are then exposed to light, both AHL and LuxR will be present, and so the lux pR promoter will be activated. When this occurs the reporter gene is expressed, as well more LuxR is produced so that the system will remain on even when the light is then taken away.
The complicated system functions in much the same way, except it contains one additional level of security, to prevent accidental activation of the system. There is an RNA lock covering the ribosome binding site in front of the second luxR in the plasmid. This way, even if the lux pR promoter is a touch leaky, no LuxR will be produced to fully activate the system.
The application below shows the schematics of both the complex and simple systems. Hovering over a part with the mouse will highlight its corresponding description in the table. Clicking on a part in the diagram will open the registry's page that desribes the part.
NOTE: if you are viewing this page with internet explorer you will have to click on the application once before you can use it
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In addition to our logic control circuit, our team has also done significant work with the protein β-agarase. β-agarase is a protein native the marine bacterial species Pseudoalteromonas atlantica . In its natural environment this bacteria uses β-agarase to degrade agar like components of seaweed into nutrient sugars. We have attempted to adapt this gene to be expressed in E. coli in such a way that its expression will activated by our light sensing system (for more on the light sensor see the light sensor section of this page). The light sensor activates the logic circuit described above which will initiate highly controlled expression of agarase.
Using the primers we designed we were able to successfully isolate the β-agarase gene from the Pseudoalteromonas atlantica genome and place it into a BioBrick plasmid (sequencing information for β-agarase is available in our final results section). However we have as yet been unable to express β-agarase in E. coli. As it stands agarase has been cloned into a biobrick vector, but because of the EcoRI and PstI sites within the gene, we are unable to attach a promoter and test the part. Directed mutagenesis will be used to silently mutate out these two sites, at which point further testing and final submission of agarase will be possible.
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Besides the laser interface, our team experimented with diffusible signaling molecules as another way of developing a highly controlled human-bacteria interface. The goal of the BioMarker project was to use an AHL induced circuit to produce images on E. coli. Ideally AHL could be selectively applied to specific parts of a plate containing our engineered cells. The cells exposed to AHL would then express our reporter gene, GFP or agarase. This would allow us to "draw" with AHL on the agar and the bacteria would then glow in the pattern applied. This project made use of the construct from the U of C 2006 iGEM project, F2620+I13504.
There were two approaches considered for this project. The first was to have an inkjet mounted on our laser plotter that would apply AHL to the agar. The second was to use a "BioMarker". That is a felt-tipped marker saturated with an AHL solution. While we have selected the latter for our project, the printer system remains a viable option that may be considered in the future. The BioMarkers were made using Fantastix blank markers by Tsukineko Corporation. The AHL was dissolved in acidified ethyl acetate to a 1uM concentration and then moved into the markers using a transfer pipette. The non-polar core of the Fantastix blank is well suited to absorbing the ethyl acetate dissolved AHL.
Early tests of the BioMarkers on regular agar plates have yielded promising results. While the agar does not readily absorb the ethyl acetate, it absorbs enough to get expression in a 3mm path around the line drawn with the thin tip type of BioMarker.
Our original plan called for applying a layer of top agar, agar that is less dense than regular agar poured over the normal agar containing our cells. However our experiments with top agar have shown that top agar is too soft to be drawn on. After a uniform lawn has been grown overnight, the 1uM BioMarker is used to draw on the agar. The response is seen after 2 hours of incubation at 37 C.
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The light sensor is an artificial gene regulation system, that translates a visible light signal into gene transcription. For our project the light sensor will activate the logic circuit described above will result in a reporter gene (agarase or GFP) being expressed by only those cells that were light activated.
The sensor originates from a light controlled gene regulatory system in the marine cyanobacterium Synechocystis sp. PCC6803, and is composed of two proteins. The first component, cyanobacterial phytochrome 1 (Cph1), is a membrane protein that serves as the light sensor. Cph1 can bind the molecule phycocyanobilin (PCB), an iron containing compound, making the complex sensitive to far red light.
Inducing gene transcription through the use of light stimulation was a very novel idea, as previously this had only been done in plants. The first working light sensor was produced in 2005 by the Austin iGEM team. To do this, they used the existing outer membrane protein (Omp) regulatory system in E.coli, another two protein system. The membrane bound sensor EnvZ is sensitive to media osmolarity; in high osmolarity EnvZ tends to phosphorylate the outer membrane protein regulator (OmpR), and in low osmolarity it de-phosphorylates the same. The mechanism for how EnvZ responds to osmolarity is unknown, but the domain for OmpR phosphorylation has been deduced. Phosphorylated OmpR (OmpR-P) regulates both of the genes ompC and ompF, both of which code for outer membrane porins. These porins are however regulated differentially, as ompC is induced by OmpR-P, and ompF is repressed. (Omp system regulation is slightly more complex than presented here, but our description good in general.)
The Austin iGEM team selected EnvZ becuase its kinase/phosphatase domain has been well studied, including studies of fusion proteins with EnvZ's kinase/phosphatase domain fused with sensory domains from other proteins (6). They created the Cph1-EnvZ fusion protein Cph8, a combination of the PCB binding domain from Cph1, and the kinase/phosphatase domain from EnvZ. Cph8 effectively bridges the gap between the light sensor and gene regulation. In light, Cph8 has little kinase activity, but in the dark the reverse is true. The ultimate result of the Austin iGEM project was an EnvZ knockout E.coli strain (CP919) expressing Cph8, HO1, PcyA, and the enzyme LacZ under control of the ompC promoter. This strain was then grown in lawns on X-gal plates, and in cells cultured in light Cph8 had little kinase activity, OmpR remained unphosphorylated, and LacZ was not expressed, leaving white cells. Cells grown in the dark had high Cph8 activity, and high levels of OmpR-P, resulting in high LacZ production, transforming X-gal into it's blue metabolite, producing darkened cells. Using transparencies, areas on a single petri plate could be exposed to light or dark, resulting in the 'coliroid' images seen in their brief communication to Nature.