Difference between revisions of "Team:Birkbeck/Conclusion"
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<p>As mentioned in the <a href="https://2015.igem.org/Team:Birkbeck/Discussion">discussion</a>, there has been success in the transduction of <i>Mycobacterium</i> cells using mycobacteriophages. These protocols require growing up cells, exposing cells to bacteriophages & conducting a plaque assay. The preliminary results from our study show that fluorescence can be detected from as early as 20-60 minutes of <i>E. coli</i> growth. This would greatly reduce the time frame of pathogen detection. The results of the interlab study also show that the protocol is adaptable to accommodate different laboratory environments.</p> | <p>As mentioned in the <a href="https://2015.igem.org/Team:Birkbeck/Discussion">discussion</a>, there has been success in the transduction of <i>Mycobacterium</i> cells using mycobacteriophages. These protocols require growing up cells, exposing cells to bacteriophages & conducting a plaque assay. The preliminary results from our study show that fluorescence can be detected from as early as 20-60 minutes of <i>E. coli</i> growth. This would greatly reduce the time frame of pathogen detection. The results of the interlab study also show that the protocol is adaptable to accommodate different laboratory environments.</p> | ||
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| − | <h3><b><u>Biobricks For | + | <h3><b><u>Biobricks For Mutagenesis</u></b></h3> |
| − | <p>The <i>stf</i> gene was successfully reconstructed with <i>orf-401</i> & <i>orf-314</i>. No mutagenesis work was carried out on <i>stf</i>. However, the biobrick is now available for mutagenesis work & future testing for changes in binding specificity.</p> | + | <p>The short tail fibre (<i>stf</i>) gene was successfully reconstructed with <i>orf-401</i> & <i>orf-314</i>. No mutagenesis work was carried out on <i>stf</i>. However, the biobrick is now available for mutagenesis work & future testing for changes in binding specificity.</p> |
| − | <p>The TFA acts as a molecular chaperone STF. The cloning of <i>tfa</i> will permit future mutagenesis work, which may facilitate the assembly of mutant STF if problems arise when mutations are introduced to <i>stf</i>. <i>tfa</i> was only cloned into the circuit & not made into a | + | <p>The tail fibre assembly (TFA) protein acts as a molecular chaperone STF. The cloning of <i>tfa</i> will permit future mutagenesis work, which may facilitate the assembly of mutant STF if problems arise when mutations are introduced to <i>stf</i>. <i>tfa</i> was only cloned into the circuit & not made into a standalone biobrick. The <i>tfa</i> gene was cloned into the circuit where it is repressed by TetR. The addition of anhydroustetracycline will alleviate the repression and therefore allow optimization of <i>tfa</i> expression to give the largest yield of recombinant virions possible.</p> |
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<h3><b><u>Biobricks for the Lytic/Lysogenic Cycle Control</u></b></h3> | <h3><b><u>Biobricks for the Lytic/Lysogenic Cycle Control</u></b></h3> | ||
| − | <p>For the biosynthesis of bacteriophage virions for our product, it is essential that the lytic & lysogenic cycles are tightly regulated in order to maximise the number of virions produced. The <i>CI</i> & <i>Cro</i> genes were successfully constructed into a circuit. With <i>CI</i> being | + | <p>For the biosynthesis of bacteriophage virions for our product, it is essential that the lytic & lysogenic cycles are tightly regulated in order to maximise the number of virions produced. The <i>CI</i> & <i>Cro</i> genes were successfully constructed into a circuit. With <i>CI</i> being constitutively expressed, lysogeny can be maintained for bacterial load to be increased in order to maximise the number of bacteriophage virions to be produced. The <i>T7-RNAP</i> is controlled under pBAD promoter which in turn stimulates the expression of <i>Cro</i>. This control can allow optimization of when to stimulate the switch from lysogeny to lytic cycle to maximise viral titre in the lysate.</p> |
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Revision as of 12:36, 18 September 2015
Conclusion
Signal Detection
As mentioned in the discussion, there has been success in the transduction of Mycobacterium cells using mycobacteriophages. These protocols require growing up cells, exposing cells to bacteriophages & conducting a plaque assay. The preliminary results from our study show that fluorescence can be detected from as early as 20-60 minutes of E. coli growth. This would greatly reduce the time frame of pathogen detection. The results of the interlab study also show that the protocol is adaptable to accommodate different laboratory environments.
Biobricks For Mutagenesis
The short tail fibre (stf) gene was successfully reconstructed with orf-401 & orf-314. No mutagenesis work was carried out on stf. However, the biobrick is now available for mutagenesis work & future testing for changes in binding specificity.
The tail fibre assembly (TFA) protein acts as a molecular chaperone STF. The cloning of tfa will permit future mutagenesis work, which may facilitate the assembly of mutant STF if problems arise when mutations are introduced to stf. tfa was only cloned into the circuit & not made into a standalone biobrick. The tfa gene was cloned into the circuit where it is repressed by TetR. The addition of anhydroustetracycline will alleviate the repression and therefore allow optimization of tfa expression to give the largest yield of recombinant virions possible.
Biobricks for the Lytic/Lysogenic Cycle Control
For the biosynthesis of bacteriophage virions for our product, it is essential that the lytic & lysogenic cycles are tightly regulated in order to maximise the number of virions produced. The CI & Cro genes were successfully constructed into a circuit. With CI being constitutively expressed, lysogeny can be maintained for bacterial load to be increased in order to maximise the number of bacteriophage virions to be produced. The T7-RNAP is controlled under pBAD promoter which in turn stimulates the expression of Cro. This control can allow optimization of when to stimulate the switch from lysogeny to lytic cycle to maximise viral titre in the lysate.