Team:SDU-Denmark/Tour32
"Design is everything. EVERYTHING!" - Paul Rand
System Design
The bacterial two-hybrid screening system is made up of two primary devices. One that is build around the T18 domain and one that is build around 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 associated protein (CAP), that binds cyclin adenosinemonophosphate (cAMP) and another which can bind the repressor protein LacI. When IPTG binds LacI, the repressor protein is inhibited.
The lac promoter is chosen in this case because, while it is inducible, it also gives a high enough expression when expressed in 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 a carbohydrate stress promoter and is induced by cAMP. This make it usable as a reporter system for our bacterial two-hybrid system that is based on the reconstitution of adenylate cyclase upon protein-protein interaction between the two proteins conjugated to T18 and T25. If using a cyaA-deficient strain, red colonies will only be seen when a protein-protein interaction occurs.
In our project we worked with the three target proteins: carbon storage regulator CsrA (BBa_K1638037), the RNase adaptive protein Yhbj (BBa_K1638038) and the RNA-binding protein Hfq (BBa_K1638039).
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]
High expression is essential for a high yield of our peptide aptamer.
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.
A 3xFLAG-tag is added to the C-terminal end 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 this device so that it contained 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. should enable affinity purification of the hTrx-based peptide aptamer.
To validate that the T18 and T25 domain constructs can be used to study protein-protein interactions, we fused leucine zippers to the two catalytic domains. Read more about this in our control experiment.
