Difference between revisions of "Team:KU Leuven/Research/Idea"

Intrigued by the patterns occuring in nature, we started our research to design possible interaction schemes and genetic circuits. Looking at specialised literature, we were able to find some interesting papers concerning the formation of patterns using Escherichia coli (E. coli). For example, Basu et al. created ring-like patterns based on chemical gradients of an acyl-homoserine lactone (AHL) signal that is synthesized by ‘sender’ cells. In receiver cells, designed genetic networks respond to differences in AHL concentrations ⁽¹⁾. Liu et al. created periodic stripes of high and low cell densities of E. coli, by inhibiting cell motility when cell density was high ⁽²⁾. Combining our own innovative and altered chemotaxis intercellular relationship with basic principles from both papers allowed us to design our own circuit which will be elucidated in the following paragraphs.

Two different cell types, called A and B, interact and create patterns. In order to achieve the desired behavior, the cells used in our experiments are derived from K12 Escherichia coli strains and have specific knockouts. Cell type A has a deletion of tar and tsr , whereas in cell type B both tar and cheZ are knocked out. Tar and Tsr are two major chemotactic receptors, guiding the cells towards nutrients. When a certain small molecule binds one of these receptors, a signalling cascade is initiated. In our specific case cell’s A deletion of both tsr and tar causes them to be insensitive to leucine (as a repellent and attractant). Cell B on the other hand, who has lost only Tar is repelled by leucine. ⁽³⁾ Upon recognition of the small molecule (in this case leucine) the receptor transduces the signal through a set of Che proteins. This group of methylesterases and phosphatases regulates the the flagellar rotation (figure 1). In E. coli, the motors turn counterclockwise (CCW) in their default state, allowing the several filaments on a cell to join together in a bundle and propel the cell smoothly forward. The interaction of phospho-CheY and a motorprotein causes the motors to switch to clockwise (CW) rotation, inducing dissociation of the filament bundle and tumbling of the cell. ⁽⁴⁾ To regulate CheY activity, a protein called CheZ removes the phosphate group from it and inhibits its activity. Cells lacking this protein are not able to swim and will tumble excessively and incessantly ⁽⁵⁾.

Both cells are transfected with only one plasmid each (Figure 1). Both plasmids contain a temperature sensitive cI repressor protein, which is constitutively expressed. This repressor is only able to bind to the lambda promoter at temperatures below a certain threshold (between 30°C and 42°C). At elevated temperatures, cI is unable to bind to the promoter region enabeling RNA polymerase to start transcription. ⁽⁶⁾ From the lambda promoter in cell A, two essential protagonists for our system are expressed: the AHL-producing enzyme LuxI and the transcription activator LuxR ⁽⁷⁾. On the contrary, cell B only contains the lambda promoter coupled to the luxR gene. In cell A, we coupled the expression of an autoaggregation factor Ag43-YFP and a leucine-producing enzyme Transaminase B to the AHL-LuxR regulated promoter. Ag43 causes cells A to stick together and form clumps ⁽⁸⁾. At the same time, Transaminase B catalyses the production of the repellent leucine ⁽⁹⁾. The repellent has no impact on cell A, since it does not contain the necessary receptors anymore. Cell B on the other hand is repelled by leucine, and contains the Lux promoter coupled to CheZ-GFP and the transcription repressor PenI. This repressor binds to the Pen-promoter ⁽¹⁰⁾, which regulates the transcription of a red fluorescent protein (RFP). The use of fluorescent fusion proteins specific for a certain state (aggregated, swimming, tumbling) facilitates the read-out of our patterns. In order to measure the concentration of the key proteins LuxI and LuxR, they were fused with a His-tag and an E-tag respectively. To ensure rapid degradation of proteins whose expression is dependent on AHL concentration, an LVA tag was fused to CheZ, GFP and RFP.

We expect that by raising the temperature, cells of type A will start to secrete AHL. This AHL will activate cells A to produce Ag43 and leucine and cells B to express CheZ-GFP and PenI. Cells A will start to form distinct spots on the agar plate and will emit yellow light (λ = 528 nm). At the same time, cells B will be repelled from cells A by leucine, the expression of PenI will repress the expression of RFP and thus the red color, whereas the expression of CheZ will enable the bacteria to swim and render them green. When they have swum too far away from cells A, the concentration of AHL decreases to levels which are too low for LuxR to bind to the promotor. When LuxR cannot bind anymore, CheZ and PenI will no longer be expressed causing the bacteria to tumble incessantly and become red again.

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