Difference between revisions of "Part:BBa K2324012"
 
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<partinfo>BBa_K2324012 short</partinfo>
 
<partinfo>BBa_K2324012 short</partinfo>
  
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This part contains the <i> fim operon </i> under control of a constitutive promoter. When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The part requires no induction, and therefore is able to continuously produce the large mass of protein encoded in the operon.  
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This part contains the <i>fim operon</i> under the control of the constitutive promoter P_J23100 (BBa_J23110). 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).  The constituent genes code for structural proteins, such as FimA which makes up the majority of the main body of a pilus, and others which support the process of pilus biogenesis. When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The part requires no induction.
  
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<!-- Add more about the biology of this part here
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We used a modular cloning strategy to build this composite part from three standard parts (https://parts.igem.org/Part:BBa_K2324016, https://parts.igem.org/Part:BBa_K2324017 and https://parts.igem.org/Part:BBa_K2324018).
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2324012 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K2324012 parameters</partinfo>
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<html>
 
<h4>Fim Operon expression</h4>
 
<h4>Fim Operon expression</h4>
  
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<p align="left">
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<figure style="border:solid black 2px;">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/e/ee/T--Exeter--MGNS.jpeg">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px" src="https://static.igem.org/mediawiki/2017/9/90/T--Exeter--Top10NS.jpeg ">
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<figcaption style="text-align:justify; margin-left:5px; margin-right:5px;">
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  <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
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  pili.
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</figcaption>
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</figure>
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</html>
  
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https://static.igem.org/mediawiki/2017/e/ee/T--Exeter--MGNS.jpeg  
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<html>
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<figure style="border:solid black 2px;">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/a/af/T--Exeter--Top10J23NS.jpeg">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/b/b3/T--Exeter--Top10AraNS.jpeg">
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<figcaption style="text-align:justify; margin-left:5px; margin-right:5px;">
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  <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
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  operon alone.
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</figcaption>
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</figure>
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</html>
  
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</p>
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<html>
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<p align="right">
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<figure style="border:solid black 2px;">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/b/bb/T--Exeter--FimBKOWT.jpeg">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/4/43/T--Exeter--FimBKOWTclose.jpeg">
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<figcaption style="text-align:justify; margin-left:5px; margin-right:5px;">
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  <b>Figure 3</b>: &#916;FimB 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 &#916;FimB 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.
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</figcaption>
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</figure>
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</html>
  
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https://static.igem.org/mediawiki/2017/9/90/T--Exeter--Top10NS.jpeg  
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<html>
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</p>
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<figure style="border:solid black 2px;">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/c/c2/T--Exeter--FimBKOj23NEW.jpeg">
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<img style="width:50%; margin-left:auto; margin-right:auto; display:block; margin-top: 10px;" src="https://static.igem.org/mediawiki/2017/1/17/T--Exeter--FimBKOPARApili.jpeg">
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<figcaption style="text-align:justify; margin-left:5px; margin-right:5px;">
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<b>Figure 4</b>: We transformed a plasmid containing the <i>fim operon</i> under control of promoters P_J23100(top)
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and P_Ara(bottom) in &#916;FimB. Both exhibit flagella on their cell surface, but the bottom electron micrograph shows a suggestion of pili.
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This suggests that the P_Ara construct was successful in synthesising pili (minus FimH).
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</figcaption>
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</figure>
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</html>
  
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<p>
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<h2>Conclusion</h2>
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<b>Figure 10 </b> On the right is an electron micrograph of an <i>E. coli </i> MG1655 cell. Pili are clearly visible on the surface of the cell. On the left is an image of Top10, which displays no pili.
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The electron micrographs presented above of MG1655 are reminiscent of those produced by Pallesen et. al. and confirm pili production in this strain as expected. Unfortunately when under control of the constitutive P_J23100 promoter pili expression is not seen.
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</p>
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<h2>References </h2>
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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.
  
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<p align="left">
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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.
  
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https://static.igem.org/mediawiki/2017/a/af/T--Exeter--Top10J23NS.jpeg
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2324012 SequenceAndFeatures</partinfo>
  
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<!-- Uncomment this to enable Functional Parameter display
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<p align="right">
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===Functional Parameters===
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<partinfo>BBa_K2324012 parameters</partinfo>
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https://static.igem.org/mediawiki/2017/b/b3/T--Exeter--Top10AraNS.jpeg
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</p>
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<p>
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<b>Figure 11 </b>Top10 with the <i>fim operon</i> under the control of promoter P_J23100 (left) and P_Ara(right), showed no visible signs of pili expression with insertion of the operon alone.
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</p>
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<p align="left">
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https://static.igem.org/mediawiki/2017/b/bb/T--Exeter--FimBKOWT.jpeg
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</p>
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<p align="right">
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https://static.igem.org/mediawiki/2017/4/43/T--Exeter--FimBKOWTclose.jpeg
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</p>
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<p>
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<b>Figure 12 </b> &#916;FimB 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 &#916;FimB with strong, peritrichous flagellar expression, but no visible signs of pili. The image on the right 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. 
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</p>
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<p align="left">
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https://static.igem.org/mediawiki/2017/4/41/T--Exeter--FimBKOj23.jpeg
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</p>
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<p align="right">
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https://static.igem.org/mediawiki/2017/1/17/T--Exeter--FimBKOPARApili.jpeg
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</p>
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<b>Figure 13 </b> We transformed a plasmid containing the <i>fim operon</i> under control of promoters P_J23100(left)
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and P_Ara(right). Both exhibit flagella on their cell surface, but the right electron micrograph shows a suggestion of pili.
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This suggests that the P_Ara construct was successful in synthesising pili (minus FimH).
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</p>
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Latest revision as of 01:23, 2 November 2017


pJ23100_FimOp

This part contains the fim operon under the control of the constitutive promoter P_J23100 (BBa_J23110). 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). The constituent genes code for structural proteins, such as FimA which makes up the majority of the main body of a pilus, and others which support the process of pilus biogenesis. When co-transformed with FimH constructs from our project, which have rhamnose-inducible promoters, modified type I pili may be produced. The part requires no induction.

We used a modular cloning strategy to build this composite part from three standard parts (https://parts.igem.org/Part:BBa_K2324016, https://parts.igem.org/Part:BBa_K2324017 and https://parts.igem.org/Part:BBa_K2324018).


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).

Conclusion

The electron micrographs presented above of MG1655 are reminiscent of those produced by Pallesen et. al. and confirm pili production in this strain as expected. Unfortunately when under control of the constitutive P_J23100 promoter pili expression is not seen.

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 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 2648
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 931
    Illegal AgeI site found at 962
  • 1000
    COMPATIBLE WITH RFC[1000]