Coding
Tse2

Part:BBa_K314200

Designed by: Matthew Coyne, Ingrid Swanson, Jesa Landis, Matthew Harger   Group: iGEM10_Washington   (2010-10-18)
Revision as of 03:13, 16 October 2019 by LLL (Talk | contribs) (→‎SZU-China 2019 iGEM team)

Toxin Tse2

A toxic protein originating from Pseudomonas aeruginosa that has been shown to arrest growth in both prokaryotic and eukaryotic cells when expressed intracelluarly. It is a substrate of the Pseudomonas aeruginosa type 6 secretion system. Tse2 has an immunity protein, Tsi2, that, when expressed in conjunction with Tse2 prevents cell death. [http://2010.igem.org/Team:Washington/Gram_Negative Find more information here on our wiki!.]

Reference showing that Tse2 is toxic, Tsi2 is the antitoxin, and that these proteins are transferred into target Gram-negative organisms via a secretion system: "A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria" [http://www.ncbi.nlm.nih.gov/pubmed/20114026]

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 196
    Illegal NgoMIV site found at 253
    Illegal NgoMIV site found at 378
  • 1000
    COMPATIBLE WITH RFC[1000]

SZU-China 2019 iGEM team

SZU-China 2019 team this year has characterized this part.

The Tse2 protein was found to be the toxin component of a toxin-immunity system and to arrest the growth of prokaryotic and eukaryotic cells when expressed intracellularly. We wanted to know whether raising the concentration of IPTG could promote the function of Tse2. To characterize this toxin Tse2, we constructed plasmid pET-28a (+) with IPTG inducible promoter and T7 terminator. Then, A growth assay has been done. Here ht115 (DE3) E. coli bacteria transformed with the toxin as well as the ones without toxin were grown for 16 hours to reach logarithmic growth phase in LB media and then moved to new LB with different concentration of IPTG to grow for more 4 hours. After that, the OD600 were measured (Fig.1).

Fig.1 OD600 of different treated samples
The results showed that toxin Tse2 could not work as expected, not to mention the effect of the IPTG concentration. However, the spread plate method results showed that this toxin could function (Fig.2) and SDS-PAGE results showed that there were Tse protein translated.
Fig.2 Growth of E. coli on Solid Media under different treated
Fig.3 SDS-PAGE of Toxin Tse2 protein
Then, we were looking for a better way to characterize toxin Tse2. SZU-China 2019 iGEM team this year aimed to develop an RNAi-based herbicide for M. micrantha. We constructed a plasmid and transformed it into E. coli ht115 (DE3), which then could transcribe hairpin siRNA. We found a G-quadruplex DNA-based, label-free and ultrasensitive strategy to detect the siRNA. Therefore, we introduced this strategy to test the function of toxin Tse2 and finally verified that Tse2 could work and this testing method was useful and reliable. In this characterization, a cDNA strand which is completely complementary to the target miRNA and partly complementary pairing with G-rich DNA was designed first. Then this cDNA can be competed off from the cDNA/G-rich DNA duplex to form a cDNA/RNA heteroduplex and release the G-rich oligo nucleotides when the target-siRNA was introduced. Recent research progress has demonstrated that, G-quadruplex DNA, a specific type of G-rich nucleic acid sequence, can be remarkably recognized by thioflavin T (ThT) with high selectivity, unlike the triplex, duplex or single-stranded forms of DNA. The fluorescence intensity of ThT exhibits a considerable increase upon binding to G-quadruplex DNA, which can be utilized as a signal reporter. The conformation of released G-rich oligonucleotides would change into G-quadruplex DNA with the presence of 2.0 mM K+. Then, the ThT remarkably recognizes and electively binds to the G-quadruplex DNA, resulting in a significant enhancement in the fluorescence signal (Fig.4).
Fig.4 Schematic representation of the direct detection of miRNAs