Parts

Overview of Parts

Our team submitted a total of 6 parts to the iGEM registry. The parts that were submitted were all components to N-acyl homoserine lactone (AHL) quorum sensing systems. The parts are all RFC10, 23, and 1000 compatible with some parts possessing other compatibilities. There are 5 basic parts that are part of the part collection and 1 composite part. The senders for the Aub, Bja, Bra, Cer, and Sin systems were submitted as part of the inductance test with the F2620 part, and compose a submitted part collection. The 1 composite part is the modular cassette (vector) for the inducer.

Design Considerations

In order to fully understand the mechanisms that facilitated AHL quorum sensing, we researched the mechanism behind AHL synthesis. The graphic below created by_____ displays the synthesis of 3-oxo-C6 AHLs. As shown below, an acyl-acyl carrier protein must react with an intermediate molecule with catalysis from the AHL synthase to produce the AHL.

Because our parts are nearly all Senders, we felt that an understanding of the AHL Receivers was also important. Utilizing the well characterized Tra system receiver, we created a 3D model of the Tra system binding its native AHL (3-oxo-C8-AHL), in which the interactions between the binding pocket and the AHL acyl tail was demonstrated. The semi-specific binding of these transcription factors gives us a better understanding on how crosstalk can occur.

From these mechanisms, we summarized the overall AHL induction mechanism below:

Quorum Sensing-F2620 Inductions

The part Bba_F2620 (found here), designed by Barry Canton and Anna Labno from MIT, is a device designed to output PoPs when LuxR is activated. This was used by the ASU team to test interactions between inducers from other quorum sensing systems. The Aub, Bja, Bra, Cer, and Sin systems stem from different organisms and their inducers were submitted as parts to the registry with the purpose of completing this induction test. With very few Senders currently found in the registry (only around 7), the addition of 5 Senders adds significant depth to the Sender pool.

Our Senders

Each Sender may also be referred to as an AHL synthase, as it produces a specific AHL. The 3D structures of the AHLs produced by the systems we examined are found in the Project Description. Our Senders from the Aub, Bja, Bra, Cer and Sin systems were submitted to the registry, while the Esa, Las, Lux, Rhl, and Rpa systems already existed in the registry. The induction test was done on a 96-well plate, which was run in a plate reader to read GFP expression levels. Aside from the controls, two different AHL concentrations were used. The AHL source that was used was filtered out via liquid-liquid extraction and then re-seeded with new cells. This allowed an 8-hour read to produce a definitive trend in the growth curve, providing information about the relationship of these Senders with F2620. Additional characterization was done via mass spectrometry, which was done on the Aub system. The Aub system was chosen as the system of interest, because of the unknown bacteria of origin and its alkane acyl tail, which are not nearly as well-characterized as the 3-oxo acyl tails from the Lux and Las systems. Also, safety information regarding potential dangers and proper disposal of AHLs are included on each Parts page.

Modular Cassette

Our 1 composite part was a modular vector designed for the incorporation of Sender sequences. This part will provide a standard cassette in which AHL synthase genes can be inserted. This part is designed to incorporate a Sender between two ribosome binding sites (RBSs), and is considered "modular," because the RFC10 prefix is added between the RBSs to allow insertion of any Sender. mCherry is also added as a visual indicator of transcription.