Difference between revisions of "Part:BBa K1846007"
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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 thus be visualised on the gel (tfa = 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 regulation of TetR, even when induced, the expression of tfa 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 therefore even in the uninduced samples 7 and 11, Figure 1, tfa bands can still be observed. Our next step is to analyse the cell pellet to determine the presence of the tfa gene in the insoluble fraction (a step we were unable to complete due to time limitations). | 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 thus be visualised on the gel (tfa = 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 regulation of TetR, even when induced, the expression of tfa 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 therefore even in the uninduced samples 7 and 11, Figure 1, tfa bands can still be observed. Our next step is to analyse the cell pellet to determine the presence of the tfa gene in the insoluble fraction (a step we were unable to complete due to time limitations). | ||
Revision as of 16:48, 19 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 produces TetR (tetracycline repressor) under control of a P(Cat) promoter.
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 thus be visualised on the gel (tfa = 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 regulation of TetR, even when induced, the expression of tfa 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 therefore even in the uninduced samples 7 and 11, Figure 1, tfa bands can still be observed. Our next step is to analyse the cell pellet to determine the presence of the tfa gene in the insoluble fraction (a step we were unable to complete due to time limitations).
http://2015.igem.org/File:BBKiGEM-SDSgel-Figure_1.jpg
http://2015.igem.org/File:BBKiGEM-SDSgel-Figure_2.jpg
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]