Difference between revisions of "Part:BBa K2324013"

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	<partinfo>BBa_K2324013 short</partinfo>		<partinfo>BBa_K2324013 short</partinfo>
−	<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). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein. This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. Harvard iGEM 2105 introduced a SpyTag at position 1 https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850004 but were unable to provide characterisation of this part. Therefore we decided to change the SpyTag for a 6x-Histidine tag to allow for easy characterisation of expression via SDS-PAGE and Western Blot. </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). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein. This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. The coding sequence is under the control of a rhamnose-inducible promoter (BBa_K902065), with the BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the <i>fim operon</i>  </p>	+
−	<p>The part acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of possible <i>E. coli </i> strains (BL21(DE3), Top10, FimB KO, FimH KO), the fusion protein can be expressed by inducing the culture at 0.6 OD with 2% rhamnose. The production of the 6xHistidine tag can be tested by the use of an SDS-PAGE and a Western blot. </p>	+	<p> The coding sequence is under the control of a rhamnose-inducible promoter (BBa_K902065), with the BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the remaining coding sequences from the <i>fim operon</i>. </p>
−	The results of an SDS-PAGE and subsequent Western blot provided bands at the corresponding molecular weights for the FimH_1His protein which also had binding affinities for an anti-His antibody. The result of a band that corresponds to a molecular weight marginally lighter than the entire intact FimH_1His evidentiated expression of the protein, and suggested that the signal peptide had been cleaved upon the proteins delivery to the cell surface membrane. This makes a case for successful export of the protein and therefore successful pilus biogenesis.	+	<p>As well as being utilised as a metal binding protein, this part also acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of <i>E. coli </i> strains (BL21(DE3), Top10, ΔFimB, and ΔFimH), the fusion protein can be expressed by inducing the culture at 0.6 OD with 2% rhamnose. The production of the 6xHistidine tag can be probed by the use of an SDS-PAGE and a Western blot. </p>
−	<html><body>	+	<p>After 24 h growth, cultures containing the P_Rha_FimH1His and wild-type strains were harvested. Cells were disrupted using BugBuster protein extraction reagent (Merck) and samples of the soluble and insoluble fractions were prepared for SDS-PAGE. Western Blots were probed with an anti-6xHis primary antibody raised in Mouse and anti-Mouse alkaline phosphatase-conjugated secondary antibody and visualised with SigmaFast BCIP/NBT.</p>
−	<img src="https://static.igem.org/mediawiki/2017/b/ba/T--Exeter--WTSDS.jpeg" height="475px" width="750px"/>	+
−	</body></html>	+
−	<b>Figure 1</b> The wells of the polyacrylamide gel contained the following, 1-10:marker,ΔFimH_22His pellet, ΔFimB_22His pellet, Top10_22His pellet, ΔFimH_22His pellet, ΔFimH_22His supernatant, Top10_22His supernatant, BL21(DE3)_22His supernatant, ΔFimB_22His supernatant and marker. The band of importance is the one at 28-30kDa which represents the FimH protein, potentially having been exported and had its signal peptide cleaved.	+	<h2>Results </h2>
−	<html><body>	+	<html>
−	<img src="https://static.igem.org/mediawiki/2017/5/5a/T--Exeter--SDS2.png" height="475px" width="750px"/>	+	<p>
−	</body></html>	+	<figure style="border:solid black 2px;">
		+	<img src="https://static.igem.org/mediawiki/2017/b/ba/T--Exeter--WTSDS.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 1</b> Image of SDS-PAGE gel stained with Coomassie Blue. The wells of the polyacrylamide gel contained the following, 1: marker, 2: ΔFimH with FimH_1His insoluble fraction, 3: ΔFimB with FimH_1His insoluble fraction, 4: Top10 with FimH_1His insoluble fraction, 5: ΔFimH with FimH_1His soluble fraction, 6: ΔFimH with FimH_1His soluble fraction, 7: Top10 with FimH with FimH_1His soluble fraction, 8: BL21(DE3) with FimH_1His soluble fraction, 9: ΔFimB with FimH_1His soluble fraction and 10: marker.
		+	</figcaption>
		+	</figure>
		+	</p>
		+	</html>
−	<b>Figure 2</b>	+	<html>
−	The wells is this gel are filled, 0-9, as follows: marker, BL21(DE3)WT pellet,Top10 WT pellet, &#916;FimB WT pellet, &#916;FimH WT pellet, BL21(DE3) WT supernatant, Top10 WT supernatant, &#916;FimB WT supernatant, &#916;FimH WT supernatant and BL21(DE3)_22His pellet.	+	<p>
−		+	<figure style="border:solid black 2px;">
−	<html><body>	+	<img src="https://static.igem.org/mediawiki/2017/5/5a/T--Exeter--SDS2.png" style="width:50%; margin-left:auto; margin-right:auto; margin-top:10px; display:block;">
−	<img src="https://static.igem.org/mediawiki/2017/0/03/T--Exeter--Westernblot.png" height="475px" width="750px"/>	+	<figcaption style="text-align: justify; margin-left: 5px; margin-right: 5px;">
−	</body></html>	+	<b>Figure 2</b> Image of SDS-PAGE gel stained with Coomassie Blue. The wells is this gel are contained the following: 0: marker, 1: WT-BL21(DE3) insoluble fraction , 2: WT-Top10 insoluble fraction, 3: WT-ΔFimB insoluble fraction, 4: WT-ΔFimH insoluble fraction, 5: WT-BL21(DE3) soluble fraction, 6: WT-Top10 soluble fraction, 7: WT-ΔFimB soluble fraction, 8: WT-ΔFimH soluble fraction and 9: BL21(DE3) with FimH_1His insoluble fraction.
		+	</figcaption>
		+	</figure>
		+	</p>
		+	</html>
		+	<html>
		+	<p>
		+	<figure style="border:solid black 2px;">
		+	<img src="https://static.igem.org/mediawiki/2017/0/03/T--Exeter--Westernblot.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>		<b>Figure 3</b>
−	The columns of the Western blot correspond to the following samples,0-9:marker,&#916;FimB_1His pellet, Top10_1His pellet,BL21(DE3)_1His pellet,BL21(DE3)_1His pellet,BL21(DE3)_1His supernatant, &#916;FimH_1His supernatant, &#916;FimB_1His supernatant, Top10_1His supernatant and marker. The bands present here demonstrate the affinity of protein with the same molecular weight as FimH with anti-His antibodies. This would imply that FimH_1His is being expressed.	+	The columns of the Western blot correspond to the following samples: 0: marker 1: ΔFimB with FimH_1His insoluble fraction, 2: Top10 with FimH_1His insoluble fraction, 3: BL21(DE3) with FimH_1His insoluble fraction, 4: BL21(DE3) with FimH_1His insoluble fraction, 5: BL21(DE3) with FimH_1His soluble fraction, 6: ΔFimH with FimH_1His soluble fraction, 7: ΔFimB with FimH_1His soluble fraction, 8: Top10 with FimH with FimH_1His soluble fraction and 9: marker.
		+	</figcaption>
		+	</figure>
		+	The images of the SDS-PAGE gels demonstrate that the cells were successfully disrupted as protein bands were seen in both soluble and insoluble fractions. However, due to the fact that the samples were crude protein extracts, the gel images do not definitively show whether FimH-1His is expressed.
		+	The Western Blot image shows bands at the corresponding molecular weights for the FimH_1His protein with (32 kDa) and without (30 kDa) the signal peptide. In the ΔFimH culture, a band is seen in the soluble fraction corresponding to the molecular weight of the un-cleaved signal peptide-FimH_1His protein. This indicates that the protein is expressed but remains in the cytoplasm of the cell. In the BL21(DE3), Top10 and ΔFimB cultures, bands are seen in the insoluble fraction corresponding to the molecular weight of the cleaved FimH_1His protein. The lack of signal peptide demonstrates that the protein has been exported from the cytoplasm and the fact that the bands only appear in the insoluble fraction suggests that FimH-1His is somehow associated with the cell membrane. It is unclear as to why export should not occur in the ΔFimH strain.
		+	</p>
		+	</html>
		+
		+	<h2>Conclusion </h2>
		+	<html>
		+	<p>The results from the Western-Blot have demonstrated successful expression of FimH_1His from the rhamnose inducible promoter in all four <i>E. coli</i> strains. The results also suggest in three out of the four strains that the protein is exported from the cell. In order to determine if FimH_1His can associate with the rest of the fim operon proteins further work is required either in BL21(DE3) (a pili producing strain) or strains which co-harbour our <a href="https://parts.igem.org/Part:BBa_K2324015">P_Ara-fim operon</a> or <a href="https://parts.igem.org/Part:BBa_K2324012">P_J23100-fim operon</a>.
		+	</p>
		+	</html>
		+	<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.
		+	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.
	<!-- Add more about the biology of this part here		<!-- Add more about the biology of this part here

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Latest revision as of 02:12, 2 November 2017

pRha_FimH_1His

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). However these two amino acids are in the pilin binding domain which may present difficulties when attempting to introduce large modifications. Harvard iGEM 2015 also introduced modifications at position 1 of the mature FimH protein. This part produces a FimH protein with a 6xHistidine tag inserted at the first amino acid position, that is the residue that remains at the N-terminus after the signal peptide has been cleaved during the membrane export process. This position is intended to improve the steric properties of the protein so as to ease the cell surface membrane export and to prevent interference with any native protein domains in the FimH which are involved in pilus biogenesis. Harvard iGEM 2105 introduced a SpyTag at position 1 https://parts.igem.org/wiki/index.php?title=Part:BBa_K1850004 but were unable to provide characterisation of this part. Therefore we decided to change the SpyTag for a 6x-Histidine tag to allow for easy characterisation of expression via SDS-PAGE and Western Blot.

The coding sequence is under the control of a rhamnose-inducible promoter (BBa_K902065), with the BBa_B0034 RBS and BBa_B0015 terminator. The part, when induced, produces a metal binding FimH protein that should involve itself in pilus biosynthesis when co-transformed with a plasmid containing the remaining coding sequences from the fim operon.

As well as being utilised as a metal binding protein, this part also acts as a reporter. It gives clear and unambiguous evidence of protein expression. Once transformed into a number of E. coli strains (BL21(DE3), Top10, ΔFimB, and ΔFimH), the fusion protein can be expressed by inducing the culture at 0.6 OD with 2% rhamnose. The production of the 6xHistidine tag can be probed by the use of an SDS-PAGE and a Western blot.

After 24 h growth, cultures containing the P_Rha_FimH1His and wild-type strains were harvested. Cells were disrupted using BugBuster protein extraction reagent (Merck) and samples of the soluble and insoluble fractions were prepared for SDS-PAGE. Western Blots were probed with an anti-6xHis primary antibody raised in Mouse and anti-Mouse alkaline phosphatase-conjugated secondary antibody and visualised with SigmaFast BCIP/NBT.

Results

The images of the SDS-PAGE gels demonstrate that the cells were successfully disrupted as protein bands were seen in both soluble and insoluble fractions. However, due to the fact that the samples were crude protein extracts, the gel images do not definitively show whether FimH-1His is expressed. The Western Blot image shows bands at the corresponding molecular weights for the FimH_1His protein with (32 kDa) and without (30 kDa) the signal peptide. In the ΔFimH culture, a band is seen in the soluble fraction corresponding to the molecular weight of the un-cleaved signal peptide-FimH_1His protein. This indicates that the protein is expressed but remains in the cytoplasm of the cell. In the BL21(DE3), Top10 and ΔFimB cultures, bands are seen in the insoluble fraction corresponding to the molecular weight of the cleaved FimH_1His protein. The lack of signal peptide demonstrates that the protein has been exported from the cytoplasm and the fact that the bands only appear in the insoluble fraction suggests that FimH-1His is somehow associated with the cell membrane. It is unclear as to why export should not occur in the ΔFimH strain.

Conclusion

The results from the Western-Blot have demonstrated successful expression of FimH_1His from the rhamnose inducible promoter in all four E. coli strains. The results also suggest in three out of the four strains that the protein is exported from the cell. In order to determine if FimH_1His can associate with the rest of the fim operon proteins further work is required either in BL21(DE3) (a pili producing strain) or strains which co-harbour our P_Ara-fim operon or P_J23100-fim operon.

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