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From 2014.igem.org

Contents 1 B2H·Design 1.1 The Signal Converter (B2H) 1.2 Three components 1.3 B2H in IgEM: 1.4 How B2H works:

B2H·Design

The Signal Converter (B2H)

Right after mutagenesis module has generate a library in the Integrated Evolution Machine (IgEM), we use the Bacterial Two-Hybrid System (B2H) to carry out the selection processes. This part of the machine should be able to tell the difference of proteins in the library and convert this protein-protein interaction signal into an output signal of reporter expression. That’s why we also call the B2H system a signal converter. This signal converter can convert different strength of protein interaction into different expression signal of the reporter gene.

Three components

There are three key components in the Bacterial Two-Hybrid System:

1. DNA binding domain;

λcI (the full-length bacteriophage lambda repressor protein, 237 amino acids)is composed of the animo-terminal DNA-binding domain and the carboxyl-terminal dimerization domain. It can bind to a specific DNA sequence called λ operator or Or2 sequence.

2. Transcription activation domain;

rpoA (α-subunit of RNA polymerase, 248 amino acids) is capable of activating the transcription of the reporter gene at downstream.

3. Reporter cassata.

The reporter cassata contain the Or2 sequence which locates at the upstream of reporter gene. It is responsible for the binding of λcI.

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The bait is fused to the C-terminal domain of λcI and the prey is fused to the N-terminal domain of rpoA. If the bait and prey interact strongly enough, rpoA will be recruited at the promoter and activate transcription of reporter gene. When the protein-protein interaction is stronger, the local concentration of rpoA will be higher, resulting in higher transcription level of the reporter gene. Finally, we are able to convert the protein-protein interaction signal into the expression signal of the reporter gene.

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B2H in IgEM:

In IgEM, the signal converter consists of three parts. The first one is plasmid pBT. The second one is M13 bacteriophage vextor. And the last one is plasmid pRPR.

1. pBT for the DNA binding domain;

pBT is a kind of plasmid that carries a low-copy p15A replication origin and confers chloramphenicol resistance. It encodes λcI fused with the protein bait on the C-terminal domain.

2.M13 bacteriophage vector for the Transcription activation domain;

M13 bacteriophage vector carries a M13 origin of replication and is gene Ⅷ deficient. It is capable of expressing the prey (representing the protein yet to be evoleved) fused to the N-terminal domain of the protein rpoA. And it is deficient in one of the M13 genes, such as pⅧ.

3. pRPT for the Reporter cassata.

In the plasmid pRPT, which uses pSBA45 backbone with a low-copy repA pSC101-derived replication origin and ampicillin resistance. We modified the lac promoter by eliminating the lacO. As a result, the modified lac promoter is not inducible by IPTG. We have also integrated a λ bacteriophage-original λ operator (Or2) at position -62. And the final promoter is called placOr2-62.

The core component of the signal converter is on pRPT, including the promoter (containing Or2 sequence) and the reporter gene. The reporter gene derives from M13 bacteriophage, such as pⅧ, which is deficient in M13 bacteriophage vector.

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How B2H works:

In IgEM, pBT and pRPR coexist in the bacteria. λcI-bait is expressed in the bacteria. Since the DNA sequence of fusion protein rpoA-target is integrated into M13 bacteriophage vector, it is expressed in the bacteria after the infection of M13 bacteriophage.

In the beginning, there is just a weak interaction between the bait protein and the target one. And the interaction can only activate correspondingly weak transcription of reporter gene- pⅧ. pⅧ protein plays a pivotal role in the release of M13 offspring and is knocked out in the genome of M13 in IgEM. Only when the pⅧ is expressed in the bacteria can the M13 which is deficient in pⅧ release from the bacteria. However, the DNA sequence of the target will be randomly mutated by the products from the mutation plasmid. Gradually, some of the targets in the bacterium will be mutated into better forms that have stronger interactions with the bait. When the protein-protein interaction is strong enough, it will enable stronger activation of pⅧ transcription. Then with the successful expression of pⅧ, M13 offspring will be generated and released from the bacteria and continue to infect the next one.