Our project aims at designing E. coli to sense its environment and act accordingly. In order to achieve this, we have categorized our projects into three main groups.
|Sensory Module||Processing Module||Lysis Module|
We have engineered two different sensors into E. coli. The first sensor is an optical sensor that detects the light present in the environment. We used Cph8, hybrid light receptor, as a optical sensor (Fig 1) . The second sensor is to a physical sensor that would sense the temperature of the environment. We used the temperature dependent ribo-switch as a physical sensor to detect the temperature of the environment (Fig 2) .
Figure 1 – Efficiency of the optical sensor was evaluated by following GFP expression in the presence and absence of light.
Figure 2: The physical sensor engineered in Chop. coli helps it to differentiate between 30⁰C and 37⁰C when GFP was fused with the temperature dependent ribo-switch.
Being aware that fermentor is always maintained at 37⁰C, we assumed that the physical sensor would help Chop. coli to differentiate its environment. However, one cannot guarantee the presence of complete darkness in the fermentor. We hypothesized if the hybrid light receptor (hybrid of light receptor and osmo regulator) could also help determine the osmolarity. The fermentor is supposed to contain sugar at a higher concentration than the environment. As expected, the light receptor could sense high sugar concentration and behave similar to darkness in the presence of high sugar concentration. This would be an advantageous feature in Chop. coli.
Figure 3. The hybrid light receptor and osmo-regulator suffers from cross talk. Response to darkness was similar to its response to high concentration of sugar. This would be advantageous for Chop. coli as either high sugar concentration or darkness present in the fermentor will keep the cells from lysis.
After sensing the environment through optical and physical sensor, Chop. coli should process the information. We introduced two different processor system: fim inversion system and cI control system.
• fim inversion system
Even though we were able to successfully construct and regulate fim inversion system using light, we were not able to control the basal fimE expression leading to the failure of this construct.
• cI control system
As expected, the cI system was able to provide an efficient control of gene expression with light despite a background expression. To reduce the background further we integrated the cI expressed from Pompc into the chromosome of E. coli. Chromosomally encoded cI reduced the background expression further (Figure 4).
Figure 4: The cI processor system was able to work efficiently independent of the phase at which the gene expression was induced.
•We used two different lysis module to in order to compare the efficiency of lysis module to eradicate the genetically modified organisms: Holin mediated cell lysis and DpnI mediated DNA damage.
•Initially we had problems in cloning both the holin and Dpn genes under PL promoter as it is a constitutive promoter and lead to rapid cell death. We finally accomplished cloning the lytic cassette using ECNR2 strain which expresses cI constitutively.
•However, neither of our lysis module was able to give us the expected result. We have already characterized individual components of our signal processing system such as the light receptor, temperature sensor and the cI processor using GFP output.
•Hence, we believe that the expression of the lysis system was not sufficient to demonstrate the expected outcome. We would like to optimize the expression of the lysis device by changing the strength of the RBS in order to prove the efficiency of our system.
•Currently, we are trying to express the lytic cassette genes from PBAD promoter. But we could not accomplish this on time.
1.Levskaya, A., et al., Synthetic biology: Engineering Escherichia coli to see light. Nature, 2005. 438(7067): p. 441-442.
2.Oleksiuk, O., et al., Thermal Robustness of Signaling in Bacterial Chemotaxis. Cell, 2011. 145(2): p. 312-321.