Difference between revisions of "Part:BBa K2324011"
 
(42 intermediate revisions by 4 users not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2324011 short</partinfo>
 
<partinfo>BBa_K2324011 short</partinfo>
  
−
<p>This part synthesises a FimH adhesin protein fused with sfGFP at its 225th amino acid residue. The coding sequence is under the control of an IPTG-inducible, T7 promoter, with a B0034 RBS and a B0015 terminator. The part, when induced, produces a fluorescent FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i>.The T7 promoter would give very strong expression and sfGFP would both give a visual indication of successful expression and folding. As a large protein, sfGFP would push the chaperone-usher pathway to its steric limits.</p>
+
<p>The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). This part produces a FimH adhesin protein fused with sfGFP (Pedelaq et al 2005) at its 225th amino acid residue, after signal peptide cleavage. Expression is under the control of an IPTG-inducible, T7 promoter (BBa_K1614000), with BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a fluorescent FimH protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i>.The T7 promoter should give very strong expression and sfGFP should both give a visual indication of successful expression and folding. As a large protein, sfGFP would push the chaperone-usher pathway to its steric limits.</p>
 +
 
 +
We have expressed this construct in BL21(DE3). Fluorescence was measured using a plate reader (Tecan) and an Amnis ImageStream ISX. Protein expression was determined via Western Blot and TEM with Immunogold labelling.
 +
 
 +
<h2>Plate reader results </h2>
 +
<html>
 
<p>
 
<p>
−
https://static.igem.org/mediawiki/2017/2/2c/T--Exeter--BL21WTplot.png
+
<figure style="border:solid black 2px;">
−
https://static.igem.org/mediawiki/2017/3/3d/T--Exeter--T7-225GFPplot.png
+
<img src="https://static.igem.org/mediawiki/2017/3/31/T--Exeter--TECANt7.png" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
 +
<b>Figure 1</b> Data from the plate reader showing fluorescence intensity for WT-BL21(DE3) and T7-FimH_225_sfGFP, average from n=3 and error bars show standard deviation of the mean.
 +
</figcaption>
 +
</figure>
 +
These results show that fluorescence from cultures containing T7-FimH_225_sfGFP is higher than WT-BL21(DE3). This shows that FimH_225_sfGFP is expressed and does give rise to fluorescence. </p>
 +
</html>
 +
 
 +
 
 +
<h2>Amnis ImageStream TMX results </h2>
 +
<html>
 +
<p>
 +
<figure style="border:solid black 2px;">
 +
<img src="https://static.igem.org/mediawiki/2017/2/2c/T--Exeter--BL21WTplot.png" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
 +
<b>Figure 2</b> Data from the Amnis ImageStream TMX show the fluorescence profile for wild-type BL21(DE3). The wild type demonstrates no significant fluorescence.
 +
</figcaption>
 +
</figure>
 
</p>
 
</p>
 +
</html>
 +
 +
<html>
 +
<p>
 +
<figure style="border:solid black 2px;">
 +
<img src="https://static.igem.org/mediawiki/2017/3/3d/T--Exeter--T7-225GFPplot.png" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
 +
<b>Figure 3</b> Data from Amnis ImageStream TMX show the fluorescence profile for BL21(DE3) with T7_FimH_225_sfGFP. This construct shows a strong fluorescent signal in the highlighted portion of the cell and it is a significant difference compared to the wild type.
 +
</figcaption>
 +
</figure>
 +
These results show that a proportion of cells in the overall culture produced strong fluorescence. This fluorescence suggests successful folding of the sfGFP which can be taken as evidence by proxy of FimH folding.
 +
</p>
 +
</html>
 +
 +
<html>
 +
<p>
 +
<figure style="border:solid black 2px;">
 +
<img src="https://static.igem.org/mediawiki/parts/5/59/T--Exeter--imagestream_ecoli02.png" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<img src="https://static.igem.org/mediawiki/parts/3/34/T--Exeter--imagestream_ecoli01.png" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
 +
<b>Figure 4</b> Data from Amnis ImageStream TMX show the fluorescence profile for individual cells of BL21(DE3) with T7_FimH_225_sfGFP. Due to the limitations arising from the magnification, the image stream can not be used to locate where the fluorescent protein resides.
 +
</figcaption>
 +
</figure>
 +
These results nonetheless support the finding that a proportion of cells in the overall culture produced strong fluorescence.
 +
</p>
 +
</html>
 +
 +
 +
<h2>Western Blot results </h2>
 +
<html>
 +
<p>
 +
<figure style="border:solid black 2px;">
 +
<img src="https://static.igem.org/mediawiki/2017/5/59/T--Exeter--western_blot_01nov.png" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
 +
<b>Figure 5</b> Image of a Western-Blot probed with Anti-GFP primary antibody raised in Mouse and Anti-mouse alkaline phosphatase conjugated secondary antibody. Lane 1 Pure GFP; Lane 2 Soluble fraction from WT-BL21(DE3); Lane 3 Insoluble fraction from WT-BL21(DE3); Lane 4 Soluble fraction from T7_FimH_225_sfGFP; and Lane 5 Insoluble fraction of T7_FimH_225_sfGFP.
 +
</figcaption>
 +
</figure>
 +
These results show that FimH-225_sfGFP is expressed and is found in the soluble fraction of the cells. Therefore we conclude that the majority of fluorescence observed is in the cytoplasm of the cell and not from FimH-225_sfGFP attached to pili.
 +
</p>
 +
</html>
 +
 +
 +
<h2>TEM with Immunogold labelling results </h2>
 +
 +
<html>
 +
<p>
 +
The Western-Blot indicated that FimH-225_sfGFP was mainly in the cytoplasm but to determine whether any proportion of expressed FimH-225sfGFP was exported from the cell and forming pili, TEM with immunogold labelling was attempted.
 +
 +
<figure style="border:solid black 2px;">
 +
<img src="https://static.igem.org/mediawiki/2017/f/f2/T--Exeter--T7immuno.jpeg" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
 +
<b>Figure 6</b> Image from TEM with Immunogold labelling. Gold particle labelling is clearly seen in the structures surrounding the cell suggesting that FimH_225_sfGFP, is exported from the cell.
 +
</figcaption>
 +
</figure>
 +
 +
</p>
 +
</html>
 +
 +
<html>
 +
<p>
 +
<figure style="border:solid black 2px;">
 +
<img src="https://static.igem.org/mediawiki/2017/0/00/T7_immuno_2.jpg" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
 +
<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
 +
<b>Figure 7</b> Image from TEM with Immunogold labelling. The gold particles appear to align with the pili on the cell surface suggesting that FimH_225_sfGFP is able to form pili on the cell surface.
 +
</figcaption>
 +
</figure>
 +
These results suggest that FimH-225_sfGFP is expressed, exported from the cell and able to attach to pili. However WT-BL21(DE3) did show some evidence of labelling of structures surrounding the cells (data not shown) therefore these results are inconclusive.
 +
</p>
 +
</html>
 +
 +
<h2>Conclusion </h2>
 +
<p>FimH_225_sfGFP has been successfully expressed from the T7 promoter as seen from the plate reader, ImageStream and Western Blots. TEM with Immunogold images suggest that at least some FimH_225_sfGFP is exported from the cell and forms pili but when compared to WT-BL21(DE3) the results are inconclusive and further work is required to properly investigate this.</p>
 +
 +
<h2>References </h2>
 +
Le Trong, I., Aprikian, P., Kidd, B. A., Forero-Shelton, M., Tchesnokova, V., Rajagopal, P., Rodriguez, V., Interlandi, G., Klevit, R., Vogel, V., Stenkamp, R. E., Sokurenko, E. V., and Thomas, W. E. (2010) Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like Sheet Twisting. Cell 141, 645–655.
  
−
<figcaption><b>Figure 4</b> These data from a flow cytometer show the fluorescence profile for wild-type (left) BL21(DE3) and BL21(DE3) with T7_FimH_225sfGFP (right).The wild type demonstrates no significant fluorescence, while same cell with the single plasmid insert shows a strong fluorescent signal in the highlighted portion of cells.</figcaption>
+
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.
−
</figure>
+
  
−
<p>We have tested this construct in BL21(DE3) using a flow cytometer to determine the fluorescence of the sample, compared to the wild type. These results show that a number of cells in the overall culture produced strong fluorescence. This fluorescence suggests successful folding of the sfGFP which can be taken as evidence by proxy of FimH folding. The result also suggests that sfGFP is able to move through the pore formed during pilus biosynthesis. </p>
+
Pédelacq, J.-D., Cabantous, S., Tran, T., and Terwilliger, T. C. (2005) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79–88.
  
 +
Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371.
  
  

Latest revision as of 02:31, 2 November 2017


T7_FimH_225sfGFP

The literature has shown that the terminal pili protein FimH (Le Trong et al 2010) can be modified by inserting heterologous sequences at position 225 and 258 (Pallesen et al 1995, Shembri et al 1999). This part produces a FimH adhesin protein fused with sfGFP (Pedelaq et al 2005) at its 225th amino acid residue, after signal peptide cleavage. Expression is under the control of an IPTG-inducible, T7 promoter (BBa_K1614000), with BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a fluorescent FimH protein that should initiate pilus biosynthesis when co-transformed with a plasmid containing the fim operon.The T7 promoter should give very strong expression and sfGFP should both give a visual indication of successful expression and folding. As a large protein, sfGFP would push the chaperone-usher pathway to its steric limits.

We have expressed this construct in BL21(DE3). Fluorescence was measured using a plate reader (Tecan) and an Amnis ImageStream ISX. Protein expression was determined via Western Blot and TEM with Immunogold labelling.

Plate reader results

Figure 1 Data from the plate reader showing fluorescence intensity for WT-BL21(DE3) and T7-FimH_225_sfGFP, average from n=3 and error bars show standard deviation of the mean.
These results show that fluorescence from cultures containing T7-FimH_225_sfGFP is higher than WT-BL21(DE3). This shows that FimH_225_sfGFP is expressed and does give rise to fluorescence.


Amnis ImageStream TMX results

Figure 2 Data from the Amnis ImageStream TMX show the fluorescence profile for wild-type BL21(DE3). The wild type demonstrates no significant fluorescence.

Figure 3 Data from Amnis ImageStream TMX show the fluorescence profile for BL21(DE3) with T7_FimH_225_sfGFP. This construct shows a strong fluorescent signal in the highlighted portion of the cell and it is a significant difference compared to the wild type.
These results show that a proportion of cells in the overall culture produced strong fluorescence. This fluorescence suggests successful folding of the sfGFP which can be taken as evidence by proxy of FimH folding.

Figure 4 Data from Amnis ImageStream TMX show the fluorescence profile for individual cells of BL21(DE3) with T7_FimH_225_sfGFP. Due to the limitations arising from the magnification, the image stream can not be used to locate where the fluorescent protein resides.
These results nonetheless support the finding that a proportion of cells in the overall culture produced strong fluorescence.


Western Blot results

Figure 5 Image of a Western-Blot probed with Anti-GFP primary antibody raised in Mouse and Anti-mouse alkaline phosphatase conjugated secondary antibody. Lane 1 Pure GFP; Lane 2 Soluble fraction from WT-BL21(DE3); Lane 3 Insoluble fraction from WT-BL21(DE3); Lane 4 Soluble fraction from T7_FimH_225_sfGFP; and Lane 5 Insoluble fraction of T7_FimH_225_sfGFP.
These results show that FimH-225_sfGFP is expressed and is found in the soluble fraction of the cells. Therefore we conclude that the majority of fluorescence observed is in the cytoplasm of the cell and not from FimH-225_sfGFP attached to pili.


TEM with Immunogold labelling results

The Western-Blot indicated that FimH-225_sfGFP was mainly in the cytoplasm but to determine whether any proportion of expressed FimH-225sfGFP was exported from the cell and forming pili, TEM with immunogold labelling was attempted.

Figure 6 Image from TEM with Immunogold labelling. Gold particle labelling is clearly seen in the structures surrounding the cell suggesting that FimH_225_sfGFP, is exported from the cell.

Figure 7 Image from TEM with Immunogold labelling. The gold particles appear to align with the pili on the cell surface suggesting that FimH_225_sfGFP is able to form pili on the cell surface.
These results suggest that FimH-225_sfGFP is expressed, exported from the cell and able to attach to pili. However WT-BL21(DE3) did show some evidence of labelling of structures surrounding the cells (data not shown) therefore these results are inconclusive.

Conclusion

FimH_225_sfGFP has been successfully expressed from the T7 promoter as seen from the plate reader, ImageStream and Western Blots. TEM with Immunogold images suggest that at least some FimH_225_sfGFP is exported from the cell and forms pili but when compared to WT-BL21(DE3) the results are inconclusive and further work is required to properly investigate this.

References

Le Trong, I., Aprikian, P., Kidd, B. A., Forero-Shelton, M., Tchesnokova, V., Rajagopal, P., Rodriguez, V., Interlandi, G., Klevit, R., Vogel, V., Stenkamp, R. E., Sokurenko, E. V., and Thomas, W. E. (2010) Structural Basis for Mechanical Force Regulation of the Adhesin FimH via Finger Trap-like Sheet Twisting. Cell 141, 645–655.

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.

Pédelacq, J.-D., Cabantous, S., Tran, T., and Terwilliger, T. C. (2005) Engineering and characterization of a superfolder green fluorescent protein. Nature Biotechnology 24, 79–88.

Schembri, M. A., Kjaergaard, K., and KLEMM, P. (1999) Bioaccumulation of heavy metals by fimbrial designer adhesins. FEMS Microbiology Letters 170, 363–371.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 451
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]