Difference between revisions of "Part:BBa K1846007"

Latest revision as of 14:49, 21 September 2015

Bacteriophage lambda tail fibre assembly (tfa) under TetR regulation

A circuit controlling the production of the tail fibre assembly (tfa) protein of bacteriophage lambda to prevent toxicity to the cell. The tfa gene operates under a TetR repressible promoter, while a second circuit, BBa_K1846003, produces TetR (tetracycline repressor) under control of a P(Cat) promoter.

With the production of the tfa protein under control of a Tet-repressible promoter, the tfa coding sequence will not be expressed due to P(TetR). However, on addition of anhydrous tetracycline (aTc), TetR will preferably bind to aTc molecule, alleviating repression of P(TetR) and allowing the initiation of transcription. The correct cloning of this BioBrick was confirmed through Sanger sequencing and agarose gel electrophoresis.

Usage and Biology

The tfa (tail fibre assembly) protein of bacteriophage lambda assists in the assembly of the stf (short tail fibre) protein into a functional short tail fibre [1],[2]. Tfa gene displays a high level of homology (~40%) with the gp38 of bacteriophage T4 [2],[3],[4].

Sequence and Features

Characterisation

We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figures 1 and 2). Under the TetR regulation, tfa gene is expressed under a tight control, and can be visualised on the gel (tfa protein = 194 aa, MW: 21602.2 Da), (Figure 1, samples 7, 8, 9 and 11, 12 and 13; Figure 2, samples 4, 5 and 6). tfa gene circuit on its own does not appear to produce tfa protein bands (Figure 1, samples 1, 2 and 3). Although the literature suggests that the protein is soluble even with high expression [1], we hypothesise the lack of bands in these samples is due to the protein being highly acidic, and thus toxic to the cell. It may be that due to the toxicity the cells that express less of the protein are selected for in these experimental conditions. Under the regulation of TetR, even when induced, the expression of the tfa gene is considerably lower than when unregulated and hence, we hypothesise, a considerable amount of soluble material can be recovered. The TetR repressor contains an LVA tag for rapid degradation and hence in the uninduced samples 7 and 11 in Figure 1, and sample 4 in Figure 2, tfa protein bands can still be observed. Our next step is to analyse the cell pellet of unregulated tfa circuit (available separately as a composite part BBa_K1846001), to determine the presence of the tfa protein in the insoluble fraction.

http://2015.igem.org/File:BBKiGEM-SDSgel-Figure_1.jpg

http://2015.igem.org/File:BBKiGEM-SDSgel-Figure_2.jpg

References

[1] Hashemolhosseini, S., Stierhof, Y. D., Hindennach, I., & Henning, U. (1996). Characterization of the helper proteins for the assembly of tail fibers of coliphages T4 and λ. Journal of Bacteriology, 178(21), 6258–6265.

[2] Montag, D., Schwarz, H., & Henning, U. (1989). A component of the side tail fiber of E. coli bacteriophage λ can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4. Journal of Bacteriology, 171(8), 4378–4384.

[3] Hendrix, R. W., & Duda, R. L. (1992). Bacteriophage lambda PaPa: not the mother of all lambda phages. Science (New York, N.Y.), 258(5085), 1145–1148. doi:10.1126/science.1439823

[4] Haggard-Ljungquist, E., Halling, C., & Calendar, R. (1992). DNA sequences of the tail fiber genes of bacteriophage P2: Evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages. Journal of Bacteriology, 174(5), 1462–1477.