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E. Colimousine

Overview

This project is destined to generate an E. Coli strain which can transport target protein along a gradient back and forth. The bacteria can bind with the target protein at one place and then, by sensing the gradient of certain amino acid, move to its destination, where it releases the protein with the help from another E. Coli strain. When this is done, it will move back to transport more target protein. In this process, four modules are necessary: binding, releasing, movement and transition.

Binding

We first need to conjugate a binding vehicle directly sensing substrate in the medium. Outer membrane protein A (OmpA)

OmpA-like transmembrane domain is an evolutionarily conserved domain of outer membrane proteins. This domain consists of an eight-stranded beta barrel. OmpA is the predominant cell surface antigen in enterobacteria found in about 100,000 copies per cell. The expression of OmpA is tightly regulated by a variety of mechanisms.

domain comes into our notice for its outstanding property that proteins linked to its C-terminal will be pushed out of the bacteria’s membrane.

Src-homology 3 (SH3) domain

Src-homology 3 (SH3) domain has high affinity for proline-rich peptides and together they can form a left-handed poly-Pro type II helix, with the minimal consensus Pro-X-X-Pro.

which has high affinity for proline-rich peptides, could act like tongs to grab any protein with a proline riich motif. For easy track, fluorescent protein mCherry was the one linked withthe binding motif (Kd=3.67μM).

SH3 binding domain (shown bound to proline-rich peptide)

OmpA transmembrane segment

The binding module eventually comprises of two proteins, namely, OmpA-SH3 protein, which functions as the binding vehicle and proline-rich containing mCherry protein, which functions as the binding substrate.

Releasing

Considering of the significance of efficient and specific release from strong binding, HIV-protease site was constructed into the binding cassette, which enables proteolytic release by HIV-protease

HIV-protease is readily available and its high efficiency, low molecular weight and high specificity matches the fine candidate

. We fused HIV-protease with OmpA and expressed it in a second E.coli strain which we immobilize at the destination, so substrate (together with SH3 domain) could be released once the E. colimousine arrives there.

Protease site between OmpA and SH3 binding domain.

Protease presented outside by OmpA fusion

Movement

Regulatory network controlling E.coli chemotaxis is one of the well-established models of signaling transduction, hence chemotaxis is most frequently used in guiding the movement of E.coli cells. Here, receptors Tar and Tar* are assembled into large clusters that are predominantly located at the poles of the bacterium could sense extracellular signals as aspartate and phenylacetic acid (PAA) respectively. The CheW protein binds to the receptors and acts as a scaffold protein to recruit CheA, whose phosphorylation is stimulated by the receptor. The phosphate is transferred to the cytoplasmic protein CheY, which controls flagellar rotation and in turn, controls the movement of E.coli. Previously, a pair of mutations in CheW* (V108M) and Tsr* (E402A) was shown to produce an orthogonal pair, where the mutated proteins interact with each other but not with wildtype Tsr or CheW. So, this can be used to toggle between activating the PAA-responsive Tar* and aspartate-responsive Tar. When we set up two gradients (PAA and aspartate) in the opposite direction, the bacteria can move forth and back.

Transition

We need to shift between two states, the binding state and the releasing state. This molecular device is well-known for its lag in phase change, which is well adapted for our application. The lag during the phase transition is appropriate for movement and transport. λ repressor is used in our project and we can see the two different states and how we shift between them from the two picture below.

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 ==Movement==
-Chemotaxis is most frequently used when it comes to the movement of E.coli cells and the regulatory network controlling E.coli chemotaxis is one of the well-studied models of signal transduction. Extracellular signals, aspartate and serine in this case, are sensed by receptors Tar and Tsr respectively. These receptors assemble into large clusters that are predominantly located at the poles of the bacterium. The CheW protein binds to the receptors and acts as a scaffold protein to recruit CheA, whose phosphorylation is stimulated by the receptor. The phosphate is transferred to the cytoplasmic protein CheY, which controls flagellar rotation and in turn, controls the movement of E.coli. Previously, a pair of mutations in CheW* (V108M) and Tsr* (E402A) was shown to produce an orthogonal pair, where the mutated proteins interact with each other but not with wildtype Tsr or CheW. So, this can be used to toggle between activating the serine-responsive Tsr* and aspartate-responsive Tar. When we set up two gradients (serine and aspartate) going in opposite direction, the bacteria can move forth and back.
+Regulatory network controlling E.coli chemotaxis is one of the well-established models of signaling transduction, hence chemotaxis is most frequently used in guiding the movement of E.coli cells. Here, receptors Tar and Tar* are assembled into large clusters that are predominantly located at the poles of the bacterium could sense extracellular signals as aspartate and phenylacetic acid (PAA) respectively. The CheW protein binds to the receptors and acts as a scaffold protein to recruit CheA, whose phosphorylation is stimulated by the receptor. The phosphate is transferred to the cytoplasmic protein CheY, which controls flagellar rotation and in turn, controls the movement of E.coli. Previously, a pair of mutations in CheW* (V108M) and Tsr* (E402A) was shown to produce an orthogonal pair, where the mutated proteins interact with each other but not with wildtype Tsr or CheW. So, this can be used to toggle between activating the PAA-responsive Tar* and aspartate-responsive Tar. When we set up two gradients (PAA and aspartate) in the opposite direction, the bacteria can move forth and back.
 ==Transition==

Team:Tsinghua/project