Difference between revisions of "Team:Birkbeck/Composite Part"
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<p>We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figure 1,2). Under the TetR regulation, tfa gene is expressed under a tight control, and can thus be visualised on the gel, 194 aa, MW: 21602.2 Da, Figure 1, samples 7,8,9 & 11,12 and 13; Figure 2, samples 4,5 and 6. Tfa gene circuit does not 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. Our next step is to analyse the cell pellet to determine the presence of the tfa gene in the insoluble fraction.</p> | <p>We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figure 1,2). Under the TetR regulation, tfa gene is expressed under a tight control, and can thus be visualised on the gel, 194 aa, MW: 21602.2 Da, Figure 1, samples 7,8,9 & 11,12 and 13; Figure 2, samples 4,5 and 6. Tfa gene circuit does not 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. Our next step is to analyse the cell pellet to determine the presence of the tfa gene in the insoluble fraction.</p> | ||
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We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figure 3). As cI protein is produced constitutively in our circuit, we expected to see a cI protein band at ca 26 kDa (cI = 237 aa, MW: 26211.8 Da). Our control E.coli 10β cells containing the cI/cro construct are showing the expected bands at ca 26 kDa, Figure 3, samples 7, 8 and 9. We attribute lack of cI bands in our T7 Express cell lysate due to T7 RNAP leakage, which would silence the cI expression. This hypothesis requires further investigation, however our preliminary results suggest the circuit could be functional.<br> | We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figure 3). As cI protein is produced constitutively in our circuit, we expected to see a cI protein band at ca 26 kDa (cI = 237 aa, MW: 26211.8 Da). Our control E.coli 10β cells containing the cI/cro construct are showing the expected bands at ca 26 kDa, Figure 3, samples 7, 8 and 9. We attribute lack of cI bands in our T7 Express cell lysate due to T7 RNAP leakage, which would silence the cI expression. This hypothesis requires further investigation, however our preliminary results suggest the circuit could be functional.<br> | ||
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<li><a href="https://2015.igem.org/Team:Birkbeck/BioBricks">Return to BioBricks overview</a></li> | <li><a href="https://2015.igem.org/Team:Birkbeck/BioBricks">Return to BioBricks overview</a></li> | ||
Revision as of 14:46, 17 October 2015
Our BioBricks
Composite Parts
tfa (tail fibre assembly) gene circuit (BBa_K1846001)
The tfa (tail fibre assembly) protein of bacteriophage Lambda assists in the assembly of the stf (short tail fibre) protein into a functional tail fibre. This part provides the gene sequence for tfa, together with a TetR repressible promoter (TetO), ribosome binding site and an rrNB T1 terminator. Presence of the gene in shipping backbone pSB1C3 was confirmed by restriction with enzymes EcoRV and PstI (Fig. 1) and by Sanger sequencing. This BioBrick has been registered as part BBa_K1846001.
a.
b. 
Fig 1. a. Predicted band size of correct (lane 1) and incorrect (lane 2) restriction. b. Observed band sizes of both samples (both the tfa circuit) match correct band sizes of 1133 and 1737 bp.
TetR circuit (BBa_K1846003)
This is a constitutive part creating a circuit for the production of the tetracyline repressor TetR, using a constitutive, medium-copy chloramphenicol promoter, ribosome binding sequence and double terminator (rrnb T1 terminator followed by a T7Te terminator).
Correct cloning of the part into the pSB1C3 shipping vector was confirmed by Sanger sequencing. This BioBrick has been registered as part BBa_K1846003.
TetR-regulated tfa (tail fibre assembly) circuit (BBa_K1846007)
To control production of the tail fibre assembly protein to prevent toxicity to the cell, we have combined the TetR circuit (BBa_K1846003) and the tfa circuit (BBa_K1846001) into a single BioBrick. With the production of the tfa protein under control of a Tet-repressible promoter, the coding sequence will remain inactive due to production of TetR. However, on addition of anhydrous tetracycline, TetR will preferably bind to this molecule, allowing the initiation of transcription. The correct cloning of this BioBrick was confirmed through Sanger sequencing and agarose gel electrophoresis (see results). This BioBrick has been registered as part BBa_K1846007.
We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figure 1,2). Under the TetR regulation, tfa gene is expressed under a tight control, and can thus be visualised on the gel, 194 aa, MW: 21602.2 Da, Figure 1, samples 7,8,9 & 11,12 and 13; Figure 2, samples 4,5 and 6. Tfa gene circuit does not 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. Our next step is to analyse the cell pellet to determine the presence of the tfa gene in the insoluble fraction.
cI-Cro circuit (BBa_K1846005)
We have created a regulatory circuit to control the lysogenic and lytic phases of bacteriophage lambda. The cI-Cro construct contains a circuit for the production - via a constitutive promoter - of the cI repressor protein (also known as the Lambda Repressor), which is responsible for keeping bacteriophage lambda in the lysogenic cycle through the cooperative binding of two repressor dimers to the DNA, repressing the Cro gene. The circuit uses a T7 promoter to drive the expression of the Cro gene in the opposite direction to the cI gene, essentially silensing the expression of the cI gene. The strength of the promoter used means that in the presence of T7 DNA polymerase, production of the Cro repressor will incapacitate production of the cI repressor and thus enables the switch from lysogenic cycle to the lytic cycle. The correct cloning of this BioBrick was confirmed through Sanger sequencing and agarose gel electrophoresis (see results). This BioBrick has been registered as part BBa_K1846005.
We characterised this construct by analysing the soluble protein fraction of the cell lysate (Figure 3). As cI protein is produced constitutively in our circuit, we expected to see a cI protein band at ca 26 kDa (cI = 237 aa, MW: 26211.8 Da). Our control E.coli 10β cells containing the cI/cro construct are showing the expected bands at ca 26 kDa, Figure 3, samples 7, 8 and 9. We attribute lack of cI bands in our T7 Express cell lysate due to T7 RNAP leakage, which would silence the cI expression. This hypothesis requires further investigation, however our preliminary results suggest the circuit could be functional.
