Part:BBa_K3333012

tac promoter (ΔlacO) -- relB -- lambda tL0 terminator

This composite part can be divided into three parts: a modified tac promoter which lacO region is deleted, relB gene and lambda t0 terminator.

Usage and Biology

The tac promoter is a functional hybrid derived from the trp and lac promoters. Derived from sequences of the trp and the lac UV5 promoters, the hybrid promoters are useful for the controlled expression of foreign genes at high levels in E. coli. It can be repressed by the lac repressor and derepressed by IPTG. Research has shown that tac promoters can be repressed by the lac repressor[1]. The tac promoter appeared to be at least 10 times more efficient than the lac UV5 promoter and at least 3 times as strong as the fully trp promoter [2]. At last hybrid tac promoter and the promoter of the lac1 gene of Escherichia coli are active in Pseudomonas [1]. However, this part is not an original tac promoter, but a modified one. The lacO operator sequence originally located in the tac promoter is deleted, after which it is no longer inhibited by lacI and can constitutively express genes.

RelB is an antitoxin which inactivate its paired toxin, relE. Toxin-antitoxin (TA) systems, which occur in bacteria and archaea, consist of a toxin and an antitoxin[3]. 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.

figure 1: the NMR structure of a mutant of E. coli RelE toxin, RelE(R81A/R83A). This relE mutant is in free state.In the free state, an alpha helix (colored with cyan) at the C terminus contacts with the beta-sheet core, forming a closed conformation.

Figure 2: the NMR structure of a mutant of E. coli RelE toxin, RelE(R81A/R83A).This is relE and its complex with an inhibitory peptide from RelB antitoxin (colored with blue). In the relE-relB complex, the cyan alpha helix turns outwards and becomes unfold, and relB contacts with the beta-sheet core instead. this helix replacement results in neutralization of a conserved positively charged cluster of relE by acidic residues from relB.[4]

In our project, relB plays an important part in rescuing the bacterial cell from relE. Bacteria carry the toxin gene in order to modify the phage. With the CRISPR/Cas9 technology, phages will undergo genetic recombination within bacteria, and their genome will carry the relE gene, thus creating a modified phage. However, the toxin protein itself can kill the bacteria, so the paired antitoxin protein is needed to antagonize the toxin and keep the bacteria safe.

Reference

[1]Bagdasarian, M. M., Amann, E., Lurz, R. & Rückert, B. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene 26, 273-282 (1984).
[2]de Boer, H. A., Comstock, L. J. & Vasser, M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A 80, 21-25, doi:10.1073/pnas.80.1.21 (1983).
[3]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
[4]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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 288
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 288
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 150
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 288
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 288
  
• 1000
  COMPATIBLE WITH RFC[1000]