Part:BBa_K3333011
HA-Up (orf 73) -- relE -- tat promoter -- HA-Down(orf 73)
This composite part can be divided into three fragments: a pair of homologous arms (Hereinafter referred to as HA) originated from Pseudomonas phage vB_PaeM_SCUT-S1, a toxin relE originated from () and a tat promoter originated from Pseudomonas aeruginosa.
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. Phage DNA was cut open by cas9 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 ().
Characterization
Part Assembly
This part is assembled with relE(https://parts.igem.org/Part:BBa_K185047), tat promoter(https://parts.igem.org/Part:BBa_K3333000) and a pair of homologous arms from Pseudomonas phage vB_PaeM_SCUT-S1(https://parts.igem.org/Part:BBa_K3333002), (https://parts.igem.org/Part:BBa_K3333003).Tat promoter and relE were synthesized and inserted into plasmid pT020. It's worthwhile to mention that the sequence of tat promoter - relE were reversely inserted into the plasmid. Plasmid with HA-Up and HA-Down that we need, named pSEVA, was previously existed in our laboratory, so we don't have to obtain the HA sequence from the phage. The fragments HA-Up, HA-Down, relE - tat promoter were cloned separately via PCR and ligated via Gibson Assembly, which forming the part HA-Up - relE - tat promoter - HA-Down. Finally this composite part was inserted into plasmid pSEVA - HA-Up - relE - tat promoter - HA-Down. The schematic diagram is shown in figure 2.
Reference
[1]Fernández-García L, Blasco L, Lopez M, et al. Toxin-Antitoxin Systems in Clinical Pathogens. Toxins (Basel). 2016;8(7):227. Published 2016 Jul 20. doi:10.3390/toxins8070227
[2]Pedersen K, Zavialov AV, Pavlov MY, Elf J, Gerdes K, Ehrenberg M. The bacterial toxin RelE displays codon-specific cleavage of mRNAs in the ribosomal A site. Cell. 2003;112(1):131‐140. doi:10.1016/s0092-8674(02)01248-5
[3]Takagi H, Kakuta Y, Okada T, Yao M, Tanaka I, Kimura M. Crystal structure of archaeal toxin-antitoxin RelE-RelB complex with implications for toxin activity and antitoxin effects. Nat Struct Mol Biol. 2005;12(4):327‐331. doi:10.1038/nsmb911
[4]Francuski D, Saenger W. Crystal structure of the antitoxin-toxin protein complex RelB-RelE from Methanococcus jannaschii. J Mol Biol. 2009;393(4):898‐908. doi:10.1016/j.jmb.2009.08.048
[5]Li GY, Zhang Y, Inouye M, Ikura M. Inhibitory mechanism of Escherichia coli RelE-RelB toxin-antitoxin module involves a helix displacement near an mRNA interferase active site. J Biol Chem. 2009;284(21):14628‐14636. doi:10.1074/jbc.M809656200
[6]Shah, N. and Naseby, D. (2014), Bioluminescence‐based measurement of viability of P seudomonas aeruginosa ATCC 9027 harbouring plasmid‐based lux genes under the control of constitutive promoters. J Appl Microbiol, 117: 1373-1387. doi:10.1111/jam.12635
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1122
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 903
Illegal PstI site found at 1122 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1122
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1122
- 1000COMPATIBLE WITH RFC[1000]
| None |