"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.

The two-hybrid system is designed in 2 plasmids. One plasmid containing the complex build around T18 and one plasmid with the T25-complex. The cells need to take up both plasmids. To be able to select for the cells which have obtain both plasmids need to have resistance for different antibiotics, in this case resistance for chloramphenicol (CML) and Kanamycin (Kan) and therefore the plasmid backbones used was standard iGEM backbones: pSB1C3 and pSB1K3 respectively.

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

The brick containing T25 consists of a PcstA promoter, a RBS and the gene encoding for red fluorescent protein (RFP) followed by two transcriptional terminators consisting each of a 64 bp stem-loop. Located downstream are a Plac promoter and RBS followed by T25 ligated to the gene of one of the 3 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.

The promoter Plac is induced by isopropyl-beta-D-1-thiogalactopyranoside (IPTG). The promoter has two binding sites: one which can bind catabolite associated protein (CAP), which is associated with glucose uptake and another which can bind the repressor protein LacI. When IPTG binds LacI, the repressor protein is inhibited. The inducible Plac promoter is chosen in this case because it can be regulated by environmental factors, but leaky enough to give a constant transcription with little to no activation. Reference: Kay Terpe. - Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Received: 30 January 2006. Revised: 18 April 2006. Accepted: 19 April 2006. Published online: 22 June 2006. Springer-Verlag 2006 (Link)

The brick containing T18 (BBa_K1638018) consists of a PLlacO-1 promoter, a ribosomal binding site (RBS), T18 domain fused to a linker, which again is fused with human thioredoxin (hTrx). The peptide library is located inside the hTrx, which is fused with 3xFLAG.

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

Figure 5: Interaction between BBa_K1638030 and BBa_K1638031.

The PLlacO-1 is a regulatory region consisting of a promoter P(L) of phage lambda with the cI binding sites replaced with lacO1. This hybrid design induce strong promotion, which can be inhibited by LacI and induced by IPTG and L-arabinose, because both molecules inhibit LacI. This promoter has been chosen for this construct because it is a very strong promoter, but at the same time it is highly repressible, therefore PLlacO-1 would give a much greater yield compared to a normal Plac promoter. 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] 3x-FLAG is an affinity tag, which can be used for detection or purification. Fr analysis of the aptamer through western blotting the 3xFLAG is good mainly because of the small size and effectiveness. 3xFLAG system is an effective system for detection of fusion proteins and because of the small size there is a low risk of altering the aptamers function and structure. Reference: Sigmar-Aldrich: 3xFLAG system. Visited: 16.09.15 - (Link)

For the purpose of making the peptide aptamer more accessible for purification and western blotting we made a modification to the brick containing T18, hTrx and the library. Upstream from hTrx the protein intein was inserted into the plasmid (BBa_K1638035). Intein is a thiol-induced self-cleavable protein, which can enables the release of a given protein fused to the C.terminal end of intein.

Intein is because of its self-cleaving ability a good addition when the peptide aptamer needs to be separated from the catalytic domain T18. This makes purification and using the aptamer much easier.

To test the design of the two-hybrid system we fused both T18 and T25 to leucine zippers (LeuZ) (BBa_K1638030 and BBa_K1638031).