Difference between revisions of "Part:BBa K2324015"

Latest revision as of 01:27, 2 November 2017

Arabinose inducible fim operon

This part contains the fim operon under the control of an inducible arabinose promoter (BBa_I13453). The operon consists of coding sequences for expression of six Fim proteins, including FimA, FimI, FimC, FimD, FimF and FimG (Le Trong et al 2010). When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The production of the operon must be initiated by inducing the culture at 0.6 OD and 2% arabinose.

Previous iGEM team, Harvard 2015 synthesised a very similar part (https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850013), however we used the modular cloning strategy to construct our complete operon from three basic parts (https://parts.igem.org/Part:BBa_K2324016, https://parts.igem.org/Part:BBa_K2324017 and https://parts.igem.org/Part:BBa_K2324018), which has resulted in scar-sequences between the parts. We also codon optimised the sequences using the IDT E. coli codon optimisation software.

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), whereas the constitutive promoter showed no suggestion of pili.

Conclusion

The electron micrographs presented above of, MG1655 and ΔFimB carrying the fim operon under control of the arabinose inducible promoter, are reminiscent of those produced by Pallesen et. al. and suggest that under control of the arabinose inducible promoter, the fim operon is expressed and pili (albeit incomplete as they are lacking FimH) are formed on the cell surface. Further work is needed to fully characterise this, particularly in combination with out modified FimH constructs. We have successfully shown a suggestion of pili using the arabinose inducible promoter and no pili production using a constitutive promoter.

References

Le Trong, I., Aprikian, P., Kidd, B. A., Thomas, W. E., Sokurenko, E. V., and Stenkamp, R. E. (2010) Donor strand exchange and conformational changes during E. coli fimbrial formation. Journal of Structural Biology 172, 380–388.

PALLESEN, L., POULSEN, L. K., CHRISTIANSEN, G., and KLEMM, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 125
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 65
Illegal BamHI site found at 2743
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1026
Illegal AgeI site found at 1057
1000
COMPATIBLE WITH RFC[1000]

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K2324015 short</partinfo>
-This part contains the <i>fim operon</i> under the control of an inducible arabinose promoter (BBa_I13453). The operon contains six Fim proteins, including FimA, FimI, FimC, FimD, FimF and FimG (Le Tong et al 2010). When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The production of the operon must be initiated by inducing the culture at 0.6 OD and 2% arabinose.
+This part contains the <i>fim operon</i> under the control of an inducible arabinose promoter (BBa_I13453). The operon consists of coding sequences for expression of six Fim proteins, including FimA, FimI, FimC, FimD, FimF and FimG (Le Trong et al 2010). When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The production of the operon must be initiated by inducing the culture at 0.6 OD and 2% arabinose.
-Previous iGEM team, Harvard 2015 synthesised a very similar part (, however we used the modular cloning strategy to construct our complete operon from three sections (https://parts.igem.org/Part:BBa_K2324016, https://parts.igem.org/Part:BBa_K2324017 and https://parts.igem.org/Part:BBa_K2324018), which has produced scar-sites between the three sections. We also codon optimised the sequences using the IDT <i>E. coli</i> codon optimisation software.
+Previous iGEM team, Harvard 2015 synthesised a very similar part (https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850013), however we used the modular cloning strategy to construct our complete operon from three basic parts (https://parts.igem.org/Part:BBa_K2324016, https://parts.igem.org/Part:BBa_K2324017 and https://parts.igem.org/Part:BBa_K2324018), which has resulted in scar-sequences between the parts. We also codon optimised the sequences using the IDT <i>E. coli</i> codon optimisation software.
@@ Line 49: / Line 49: @@
   <b>Figure 4</b>: We transformed a plasmid containing the <i>fim operon</i> under control of promoters P_J23100(top)
   and P_Ara(bottom) in &#916;FimB. 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), whereas the constitutive promoter showed no suggestion of pili.
   </figcaption>
 </figure>
 </html>
-<h4>Conclusion</h4>
+<h2>Conclusion</h2>
-The electron micrographs presented above suggest that under control of the arabinose inducible promoter, the fim operon is expressed and pili (albeit incomplete as they are lacking FimH) are formed on the cell surface. Further work is needed to investigate further, particularly in combination with out modified FimH constructs.
+The electron micrographs presented above of, MG1655 and &#916;FimB carrying the fim operon under control of the arabinose inducible promoter, are reminiscent of those produced by Pallesen et. al. and suggest that under control of the arabinose inducible promoter, the fim operon is expressed and pili (albeit incomplete as they are lacking FimH) are formed on the cell surface. Further work is needed to fully characterise this, particularly in combination with out modified FimH constructs. We have successfully shown a suggestion of pili using the arabinose inducible promoter and no pili production using a constitutive promoter.
 <h2>References </h2>
 Le Trong, I., Aprikian, P., Kidd, B. A., Thomas, W. E., Sokurenko, E. V., and Stenkamp, R. E. (2010) Donor strand exchange and conformational changes during E. coli fimbrial formation. Journal of Structural Biology 172, 380–388.
+PALLESEN, L., POULSEN, L. K., CHRISTIANSEN, G., and KLEMM, P. (1995) Chimeric Fimh Adhesin of Type-1 Fimbriae - a Bacterial Surface Display System for Heterologous Sequences. Microbiology 141, 2839–2848.