Part:BBa_K3333018
HA-Up(orf53) - relE - tat promoter - HA-Down(orf53)
Usage and Biology
Toxin-antitoxin (TA) systems, which occur in bacteria and archaea, consist of a toxin and an antitoxin[1]. A toxin could inhibit cell growth or cause cell death, while an antitoxin could combine with its paired toxin specifically and rescue cells from being poisoned. According to the reaction mechanism, TA systems can be classified into five groups (type I to V). RelE/B, the TA system we use, belongs to type II TA system. RelE inhibits protein synthesis by cleaving mRNA codons in the ribosomal A site in a sequence specific way with preference for the stop codon UAG[2]. Although the species and active parts varies, several studies of the three-dimensional structure of relE have shown that an Arg residue at the active site plays a crucial role in the functioning of relE, and if the Arg was mutated to other amino acids[3][4], the activity of relE decreased significantly[5]. Antitoxin suppress the activity of toxin by directly binding to toxin protein.
Tat promoter is a constitutive promoter from Pseudomonas aeruginosa PAO1. Shah and Naseby constructed plasmids carrying lux genes which are under the control of constitutive promoters and tested the strength of five different constitutive promoters (Plpp, Ptat, PlysS, PldcC, Pspc) with the method of bioluminescence-based measurement[6]. They found that Promoter strength decreased in the order of Plpp > Ptat > PlysS > PldcC > Pspc during exponential phase whilst Ptat was stronger than Plpp during stationary phase. Stationary phase was observed from 12 h for all the strains and remained constant up to 48 h.
In our project, tat promoter is designed to be recombined to phage genome which will be injected into a P. aeruginosa cell. Considering relE gene need express continuously and efficiently when injected into bacteria, a constitutive promoter should be used rather than regulatory promoters. However, the function of the very one promoter varies in different species, so it’s necessary to use a proper constitutive promoter which can work efficiently in P. aeruginosa. Thanks to Shah and Naseby’s work, tat promoter fit our requirement best. Controlled by the tat promoter, relE gene get expressed in a maximum level, making sure to kill the bacteria.
HA participate in gene recombination between donor plasmid and phage genomes. Some fragments of phage DNA are selected as homologous arms, which are divided into HA-up and HA-down, and these two sequences are connected to the upstream and downstream of tat promoter-RelE respectively by overlap PCR. In this part, the phage genome's Open reading frame No. 73 (orf53) was selected as the homologous arm. Orf53 is predicted to code for the holin protein which play an important role in host lysis. Since we aim to design an non-host-lysis phage that kill the bacteria with toxin protein, orf53 is supposed to be knocked out. So, we constructed a composite part "HA-Up(orf53) - relE -tat promoter - HA-Down(orf53)".With CRISPR-Cpf1 and lambda-red system, relE, a toxin protein, and tat promoter, its constitutive promoter, will replace holin gene. In this way, holin gene could be replaced by relE-tat promoter. Phage DNA was cut open by Cpf1 protein to form double-strand break, and the incision just separated HA-up and HA-down. Gene of interest could be transferred to phage DNA from donor plasmid via forming Holliday Junction and the participance of λ-red. For more information about homologous recombination, please turn to [1].
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 912
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 180
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 98
Illegal SapI.rc site found at 415
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