Difference between revisions of "Part:BBa K3333011"

Latest revision as of 12:19, 5 October 2020

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 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.

Figure 1:Crystal structure of archaeal toxin-antitoxin RelE-RelB complex (id in PDB: 1WMI). The green one represents relE while the blue one represents relB. The required group for the relE is Arg85, which is marked in yellow in the figure. The residues marked in red are Arg40, Leu48, Arg58 and Arg65. They play a modest role in the toxin's activity. relB wraps around the molecular surface of a RelE.)

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 (orf73) was selected as the homologous arm. Orf 73 is predicted to express an unnecessary protein, which means no side effect will arise when it is inserted by foreign DNA. 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 ().

Figure 2: schematic diagram of recombination in the case of orf73

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. HA-Up and relE - tat promoter were assembled via overlap PCR. Then HA-Up - relE - tat promoter, HA-Down and plasmid pIG20NO3 were pur together via enzymatic ligation. The schematic diagram is shown in figure 3.

Figure 3: schematic diagram of part assembly of K3333011

Through DNA electrophoresis, we could verify whether the part is assembled successfully or not. The gel map shown below contains several bands, including its fragments: HA-Up, relE - tat promoter, HA-Down, and primary products HA-Up - relE - tat promoter. It also contain the bands indicating the whole part and the part digested by enzyme. All results show that the part was well assembled.

Figure 4: DNA electrophoresis gel map of K3333011 and its basic parts.

Electrotransfer

Elextrotransfer is an effective way to transfer plasmids into cells. Through high intensity electric field to improve membrane permeability, cells could absorb foreign nucleotides, DNA, RNA, proteins or virus particles. The thought of electrotransfer are as followed: preparation of competent cells with 300mM sucrose solution - mix 1ug plasmids to be transferred with 50ul cell suspension - electrotransfer - Apply cell suspension to plates with particular antibiotics - screen for cells containing aimed plasmids via colony PCR.

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