Difference between revisions of "Part:BBa K2324012:Design"
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<h4>Fim Operon expression</h4> | <h4>Fim Operon expression</h4> | ||
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https://static.igem.org/mediawiki/2017/e/ee/T--Exeter--MGNS.jpeg | https://static.igem.org/mediawiki/2017/e/ee/T--Exeter--MGNS.jpeg | ||
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https://static.igem.org/mediawiki/2017/9/90/T--Exeter--Top10NS.jpeg | https://static.igem.org/mediawiki/2017/9/90/T--Exeter--Top10NS.jpeg | ||
</p> | </p> | ||
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| − | <b>Figure | + | <b>Figure 1 </b> Top is an electron micrograph of an <i>E. coli </i> MG1655 cell. Pili are clearly visible on the surface of the cell. Bottom is an image of Top10, which displays no pili. |
</p> | </p> | ||
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https://static.igem.org/mediawiki/2017/a/af/T--Exeter--Top10J23NS.jpeg | https://static.igem.org/mediawiki/2017/a/af/T--Exeter--Top10J23NS.jpeg | ||
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https://static.igem.org/mediawiki/2017/b/b3/T--Exeter--Top10AraNS.jpeg | https://static.igem.org/mediawiki/2017/b/b3/T--Exeter--Top10AraNS.jpeg | ||
</p> | </p> | ||
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| − | <b>Figure | + | <b>Figure 2 </b>Top10 with the <i>fim operon</i> under the control of promoter P_J23100 (top) and P_Ara(bottom), showed no visible signs of pili expression with insertion of the operon alone. |
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https://static.igem.org/mediawiki/2017/b/bb/T--Exeter--FimBKOWT.jpeg | https://static.igem.org/mediawiki/2017/b/bb/T--Exeter--FimBKOWT.jpeg | ||
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https://static.igem.org/mediawiki/2017/4/43/T--Exeter--FimBKOWTclose.jpeg | https://static.igem.org/mediawiki/2017/4/43/T--Exeter--FimBKOWTclose.jpeg | ||
</p> | </p> | ||
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| − | <b>Figure | + | <b>Figure 3 </b> ΔFimB should not, theoretically, produce pili. The regulatory gene FimB has been knocked out, and so the operon has effectively been switched off. These electron micrographs show wild type ΔFimB with strong, peritrichous flagellar expression, but no visible signs of pili. The image on the bottom shows little evidence of pili or flagella connected to the cell surface, which suggests that the negative staining technique can be damaging to these structures and could cause detachment and fragmentation. |
</p> | </p> | ||
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| − | https://static.igem.org/mediawiki/2017/ | + | https://static.igem.org/mediawiki/2017/c/c2/T--Exeter--FimBKOj23NEW.jpeg |
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https://static.igem.org/mediawiki/2017/1/17/T--Exeter--FimBKOPARApili.jpeg | https://static.igem.org/mediawiki/2017/1/17/T--Exeter--FimBKOPARApili.jpeg | ||
</p> | </p> | ||
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| − | <b>Figure | + | <b>Figure 4 </b> We transformed a plasmid containing the <i>fim operon</i> under control of promoters P_J23100(top) |
| − | and P_Ara( | + | and P_Ara(bottom) in ΔFimB. Both exhibit flagella on their cell surface, but the bottom electron micrograph shows a suggestion of pili. |
This suggests that the P_Ara construct was successful in synthesising pili (minus FimH). | This suggests that the P_Ara construct was successful in synthesising pili (minus FimH). | ||
</p> | </p> | ||
Latest revision as of 23:23, 1 November 2017
pJ23100_FimOp
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2648
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 931
Illegal AgeI site found at 962 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
The operon sequence had to be divided up for synthesis (due to its length) before being ligated together. The natural transcriptional machinery of the operon had to be preserved throughout this cloning process.
Source
Part BBa_K1850012 was the source for the sequence of the operon for the CDS. Parts BBa_B0034 and BBa_B0015 were the ribosome binding site and terminator, respectively, found on the registry. The source for the J23100 promoter was part BBa_J23100.
Fim Operon expression
Figure 1 Top is an electron micrograph of an E. coli MG1655 cell. Pili are clearly visible on the surface of the cell. Bottom is an image of Top10, which displays no pili.
Figure 2 Top10 with the fim operon under the control of promoter P_J23100 (top) and P_Ara(bottom), showed no visible signs of pili expression with insertion of the operon alone.
Figure 3 ΔFimB should not, theoretically, produce pili. The regulatory gene FimB has been knocked out, and so the operon has effectively been switched off. These electron micrographs show wild type ΔFimB with strong, peritrichous flagellar expression, but no visible signs of pili. The image on the bottom shows little evidence of pili or flagella connected to the cell surface, which suggests that the negative staining technique can be damaging to these structures and could cause detachment and fragmentation.
Figure 4 We transformed a plasmid containing the fim operon under control of promoters P_J23100(top) and P_Ara(bottom) in ΔFimB. Both exhibit flagella on their cell surface, but the bottom electron micrograph shows a suggestion of pili. This suggests that the P_Ara construct was successful in synthesising pili (minus FimH).