Difference between revisions of "Part:BBa K1846007"
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A circuit controlling the production of the tail fibre assembly (tfa) protein of bacteriophage lambda. The ''tfa'' gene operates under a TetR repressible promoter, while a second circuit, [https://parts.igem.org/Part:BBa_K1846003 BBa_K1846003], produces TetR (tetracycline repressor) under control of a P(Cat) promoter. | A circuit controlling the production of the tail fibre assembly (tfa) protein of bacteriophage lambda. The ''tfa'' gene operates under a TetR repressible promoter, while a second circuit, [https://parts.igem.org/Part:BBa_K1846003 BBa_K1846003], produces TetR (tetracycline repressor) under control of a P(Cat) promoter. | ||
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| + | ===Usage and Biology=== | ||
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| + | 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]. | ||
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| + | ===Functional Parameters=== | ||
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| + | <partinfo>BBa_K1846007 parameters</partinfo> | ||
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). We attribute the lack of bands in these samples to protein overexpression and aggregation into insoluble fraction. 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 [https://parts.igem.org/Part:BBa_K1846001 BBa_K1846001]), to determine the presence of the tfa protein in the insoluble fraction. | 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). We attribute the lack of bands in these samples to protein overexpression and aggregation into insoluble fraction. 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 [https://parts.igem.org/Part:BBa_K1846001 BBa_K1846001]), to determine the presence of the tfa protein in the insoluble fraction. | ||
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[[File:BBKiGEM-SDSgel-Figure_2.jpg|400px|thumb|left|]] | [[File:BBKiGEM-SDSgel-Figure_2.jpg|400px|thumb|left|]] | ||
| + | ===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 | |
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| − | + | [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. | |
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Revision as of 18:29, 20 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. 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.
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].
Functional Parameters
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). We attribute the lack of bands in these samples to protein overexpression and aggregation into insoluble fraction. 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.