B2H

The Signal Converter (B2H):

In order to operate the Integrated Evolution Machine (IgEM), we have constructed a signal converter in our machine. This signal converter can transform an input signal of protein-protein interaction into an output signal of reporter expression. In fact, it is the Bacterial Two-Hybrid System (B2H). (reference)

Here is how it functions.

The Or2 sequence contains the DNA binding domain of λcI (the full-length bacteriophage lambda repressor protein, 237 amino acids) and locates at upstream of the promoter. It is responsible for the binding of λcI. λcI is composed of the animo-terminal DNA-binding domain and the carboxyl-terminal dimerization domain. And rpoA (α-subunit of RNA polymerase, 248 amino acids) is capable of activating the transcription of the reporter gene at downstream.

(the picture should be affected) Then the protein bait is fused to the C-terminal domain of λcI and the protein target is fused to the N-terminal domain of rpoA. The target protein is the one that is going to be mutated to interact with the bait protein. If the protein bait and target interact strongly enough, more 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.

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.

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.

M13 bacteriophage vector carries a M13 origin of replication and confers kanamycin resistance. It is capable of expressing the target protein fused to the N-terminal domain of the protein rpoA. And it is deficient in one of the M13 genes, such as pⅧ.

In the plasmid pRPR, which uses pSBA45 backbone with a low-copy repA pSC101-derived replication origin and ampicillin resistance, we have 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 pRPR, 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. (M13)

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.

Team:SYSU-China/file/Project/Design/B2H.html

From 2014.igem.org

Contents

B2H

The Signal Converter (B2H):

Here is how it functions.

B2H in IgEM:

How B2H works: