"Design is everything. EVERYTHING!" - Paul Rand

System Design

Figure 1: Target Device: Target protein fused to T25 through a flexible linker. Device also contains a cAMP-induced RFP reporter system.

Figure 2: Peptide aptamer Device: Peptide aptamer-scaffold fused to the T18 domain through a flexible linker.

The bacterial two-hybrid screening system is made up of two primary devices. One containing the T18 domain and containing the T25 domain.

Our device with the T25 domain is our target device. It contains a target conjugated to the T25 domain through a flexible linker. The gene is controlled by the lac promoter, Plac lac promoterThe lac promoter is inducible by isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The promoter has two binding sites: one which can bind catabolite activator protein (CAP), that binds cyclic adenosine monophosphate (cAMP) and another which can bind the repressor protein LacI. When IPTG binds LacI, the repressor protein is inhibited. . The lac promoter is chosen because even when not induced, it still has a sufficient expression when placed on high-copy plasmids such as pSB1C3 and pSB1K3.

This device also contains a generator of red fluorescent protein (RFP) controlled by the cstA promoter. PcstA is induced by cAMP, which makes it usable as a reporter system for our bacterial two-hybrid system. When using a cyaA-deficient strain, red colonies will form when a protein-protein interaction occurs.

In our project we worked with the three target proteins: carbon storage regulator A, CsrA, (BBa_K1638037), the RNase adaptive protein, Yhbj, (BBa_K1638038) and the RNA-binding protein, Hfq. (BBa_K1638039).

Figure 3: Modified peptide aptamer Device: Peptide aptamer-scaffold fused to the T18 domain through intein and a flexible linker.

Our device with T18 domain is our peptide aptamer device. It contains the human thioredoxin (hTrx)-based peptide aptamer conjugated to the T18 domains through a flexible linker. The device is controlled by the strong hybrid promoter PLlacO-1. Reference: Lutz R, Bujard H. Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 1997 Mar 15;25(6):1203-10
[PubMed]

The hTrx-scaffold contains a XhoI restriction site that enables insertion of a random nucleotide library. The resctrion site is situated in the active site of hTrx leaving it functionless when a library is inserted. The design of this peptide aptamer is inspired by Borghouts C. et al.. Reference: Borghouts C, Kunz C, Delis N, Groner B. Monomeric Recombinant Peptide Aptamers Are Required for Efficient Intracellular Uptake and Target Inhibition. Molecular Cancer Research. 2008;6(2):267-81.
[Mol Canc Res] They modified this scaffold for optimal use. This includes mutations that prevents multimerization of the protein and induces flexibility of the inserted peptide aptamer. See BBa_K1638014 for further details.

Figure 4: Control device: Leucine zippers fused to the T18 and T25 domains of the adenylate cyclase CyaA.

A 3xFLAG-tag is added to the C-terminal of the scaffold. This affinity tag can be used for detection and/or purification purpo ses. Reference: Sigmar-Aldrich: 3xFLAG system. Visited: 16.09.15 -

We also made another version of the device containing intein. Intein Intein Intein is a thiol-induced self-cleavable protein that enables the release of a C-terminal fused protein. It also contains a chitin-binding domain, which is an affinity-tag that enables affinity purification on a chitin column. First the target protein fused to intein is loaded and washed on the chitin column. Using a thiol reagent, like dithiothreitol (DTT), an on-column cleavage is induced and the target protein is released. enables affinity purification of the hTrx-based peptide aptamer and self-cleavaging ability, thus releasing the peptide aptamer from the purification column.

To validate that the T18 and T25 domain constructs can be used to study protein-protein interactions, we fused leucine zippers Leucin Zippers Interaction between the leucine zippers will induce formation of cAMP. to the two catalytic domains. Read more about this in our control experiment.