Difference between revisions of "Part:BBa K4491007"

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pBAD_AP

This gene encodes an improved version of the wild-type araBAD promoter (nicknamed PBAD), an inducible promoter controlled by araC protein. The native PBAD is part of the araBAD operon, which is responsible for regulating arabinose metabolism in E.coli.

The Cambridge 2022 Team had intended to use the araBAD promoter to build an antithetic feedback circuit that demonstrates robust perfect adaptation (RPA). However, we were concerned that wild-type PBAD is relatively large, topping at around 300 bp, which increases the bulkiness of our Level-2 construct and reduces efficiency. Also, P_BAD, by nature, is a relatively leaky and medium-strength promoter, therefore, lots of araC is needed to fully reach maximal activation. However, overexpression of araC is toxic to cells, and thus can drastically impede performance of the circuit. we therefore thought it would be an interesting Part Improvement project to redesign the existing wild-type promoter. Our initial goal was to engineer a PBAD with minimal length, but shows both lower leakiness and higher maximal activity. To achieve this, we did intensive literature search for prior optimization strategies. To understand the rationales, we also gathered information on the regulatory mechanism of the promoter, which was presumably only prevalent in papers from the 1970s.

Usage and Biology

The exact definition of the araBAD promoter varies ambiguously between sources. Historically, PBAD only refers to a short segment upstream of the +1 transcription start site (referred to as the core promoter), containing the -35 and -10 boxes. However, as a complete promoter Part, the regulatory region further upstream is also included. In the following discussion, our definition of P_BAD refers to the whole sequence consisting of both the regulatory sequence and the core promoter.

Figure 1: Schematic diagram of the araBAD operon and its upstream regulatory region. The araBAD promoter regulates the araBAD operon and is controlled by araC - a regulatory protein known for its “love-hate” mechanism of action. The upstream regulatory region consists of various protein binding sites - araI1, araI2, araO1 and araO2 can be occupied by araC. Between araI1 and araO1 also lies a CAP binding site, which recruits Catabolite receptor protein for transcription activation. The spacing between araO1 and O2 is noticeably large, reaching nearly 210 bp, which, unsurprisingly, is responsible for the overall bulkiness of the promoter. Interestingly, a region within this spacer contains a promoter of araC gene, which runs in the opposite direction to the araBAD operon. This promoter is regulated by araO1 just downstream. The araC protein therefore not only regulates the araBAD operon but also controls its own production by binding to araO1 region (negative autoregulation). In the absence of L-arabinose, transcription is repressed by the action of two araC molecules binding to the structure. One araC binds to araI1 and the other binds to araO2 further upstream. The dimerization domain confers high affinity, bringing the two protein molecules closer to dimerize, essentially creating a DNA loop as a result. This looping mechanism prevents any sigma factors, RNA Polymerase or CRP from being recruited, thus repressing transcription. In the presence of L-arabinose, binding of the sugar molecule to the arabinose-binding domain triggers a conformational change in the DNA-binding domain of araC, which reduces its affinity to distal araO2 site. This makes the protein more favourable to bind to araI2, just downstream of araI1. Therefore, the dimerized complex is formed at araI1-araI2 site instead of araO2-araI1, which breaks the loop and hence activates transcription. The dimer araC also “nudges” the RNA Polymerase for enhanced transcription.

(A) Adherens junctions, in the form of adhesion belts, between epithelial cells in the small intestine. The beltlike junction encircles each of the interacting cells. Its most obvious feature is a contractile bundle of actin filaments running along the cytoplasmic surface of the junctional plasma membrane. (B) Some of the molecules that form an adherens junction. The actin filaments are joined from cell to cell by transmembrane adhesion proteins called cadherins. (Alberts B. Molecular Biology of the Cell. 6th ed. New York: Garland Science; 2015)

Cadherins are a diverse family of adhesion molecules that fulfil these requirements. They are present in all multicellular animals whose genomes have been analysed. Other eukaryotes, including fungi and plants, lack cadherins, and they are also absent from bacteria and archaea. Cadherins therefore seem to be part of the essence of what it is to be an animal.

The cadherins take their name from their dependence on Ca²⁺ ions: removing Ca²⁺ from the extracellular medium causes adhesions mediated by cadherins to come apart. The first three cadherins to be discovered were named according to the main tissues in which they were found:

- E-cadherin is present on many types of epithelial cells;

- N-cadherin on nerve, muscle and lens cells;

- P-cadherin on cells in the placenta and epidermis.

All are also found in other tissues. These and other classical cadherins are closely related in sequence throughout their extracellular and intracellular domains.

Binding between cadherins is generally homophilic. This means cadherin molecules of a specific subtype on one cell bind to cadherin moleculs of the same or closely related subtype on adjacent cells. All members of the superfamily have an extracellular portion consisting of several copies of the extracellular cadherin (EC) domain. Homophilic binding occurs at the N-terminal tips of the cadherin molecules - the cadherin domains that lie furthest from the membrane. These terminal domains each form a knob and a nearby pocket, and the cadheirn molecules protruding from opposite cell membranes bind by insertion of the knob of one domain into the pocket of the other.

Figure 2: Molecular Model of E-cadherin

After processing in the late Golgi, E-cadherin contains five EC domains. The outermost EC domain forms homophilic connections with the equivalent domain of E-cadherin on the neighbouring cell. The stability of E-cadherin depends on the presence of Ca²⁺ in the extracellular space. (Alberts B. Molecular Biology of the Cell. 6th ed. Figure 19-6 New York: Garland Science; 2015)

Each cadherin domain forms a more-or-less rigid unit, joined to the next cadherin domain by a hing. Ca²⁺ ions bind to sites near each hinge and prevent it from flexing, so that the whole string of cadherin domains behaves as a rigid and slightly curved rod. When Ca²⁺ is removed, the hinges can flex, and the structure becomes floppy. At the same time, the conformation at the N-terminus is thought to change slightly, weakening the binding affinity for the matching cadherin molecule on the opposite cell.

The cadherins form homodimers in the plasma membrane of each interacting cell. The extracellular domain of one cadherin dimer binds to the extracellular domain of an identical cadherin dimer on the adjacent cell. The intracellular tails of the cadherins bind to anchor proteins that tie them to actin filaments. These anchor proteins include α-catenin, β-catenin, γ-catenin (also called plakoglobin), α-actinin, and vinculin.

UCL iGEM 2017 believes that cadherin proteins will be powerful modulators for efficient tissue engineering. We therefore investigated first the properties of one classical cadherin (E-cadherin, BBa K2332312) and then tried to make it light-responsive.

For more information on cell-cell junctions and cadherins see Alberts B., Molecular Biology of the Cell. 6th ed., Ch.19, New York: Garland Science; 2015.

E-Cadherin Entries in the Registry