Difference between revisions of "Part:BBa K1739003"
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<partinfo>BBa_K1739003 short</partinfo>
 
<partinfo>BBa_K1739003 short</partinfo>
  
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This BioBrick contains the constitutive promoter [https://parts.igem.org/Part:BBa_J23104 BBa_J23104] and uses the pSB1C3 backbone. It consists of three genes, a csgA signal sequence, Sup35-NM ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1739002 BBa_K1739002]), and Cytochrome <i>b</i><sub>562</sub> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1739002 BBa_K1739001]). The bipartite CsgA signal sequence targets the Sec protein export pathway followed by the endogenous curli export system of E.coli allowing our protein to be easily exported into an external medium (Sivanathan and Hochschild, 2012; Sivanathan and Hochschild, 2013). Sup35-NM is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid (Frederick et al., 2014; Glover et al. 1997). This has previously been discussed by Tessier and Lindquist (2009) who show that two beta-sheets bond together in a self-complimenting ‘steric zipper’ that excludes water, leaving a highly stable parallel beta-sheet with one molecule every 4.7Å. The particular advantage of using Sup35-NM is that in its native state Sup35p has two functional domains, the N and C terminal, separated by the highly charged M domain (Frederick et al., 2014; Glover et al. 1997; Wickner et al., 2007). Thus facilitating the removal of one functional domain in order to add our own functional protein, in this case cytochrome b562 to form a fusion protein.
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This BioBrick encodes for the Envirowire fusion protein. It contains three segments, CsgA signal sequence, Sup35NM (https://parts.igem.org/wiki/index.php?title=Part:BBa_K1739000 BBa_K1739000] and https://parts.igem.org/wiki/index.php?title=Part:BBa_K1739002 BBa_K1739002]) and Cytochrome <i>b</i><sub>562</sub> ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K1739001 BBa_K1739001]) The bipartite CsgA signal sequence targets the Sec protein export pathway followed by the endogenous curli export system of E.coli allowing our protein to be easily exported into an external medium (Sivanathan and Hochschild, 2012; Sivanathan and Hochschild, 2013). Sup35NM is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid (Frederick et al., 2014; Glover et al. 1997). This has previously been discussed by Tessier and Lindquist (2009) who show that two beta-sheets bond together in a self-complimenting ‘steric zipper’ that excludes water, leaving a highly stable parallel beta-sheet with one molecule every 4.7Å. The particular advantage of using Sup35-NM is that in its native state Sup35p has two functional domains, the N and C terminal, separated by the highly charged M domain (Frederick et al., 2014; Glover et al. 1997; Wickner et al., 2007). ). Here, with the removal of the functional C domain allows us to add our own functional protein, in this case cytochrome b562, to form a fusion protein. We chose Cytochrome b562 as the electron carrier to make our amyloid conductive. The structure of cytochrome b562 consists of a single 24kDa subunit containing four nearly parallel alpha helices (Fujiwara, Fnkumori, and Yamanaka, 1993; Mathews et al., 1979). B-type cytochromes are a favourable choice because heme binds in a non-ionic fashion to the two ligands Methionine-7 and Histidine-106 (Xavier et al., 1978). Heme binding has been shown to occur in both the native protein and the denatured protein, although the latter exhibits a modest affinity with a dissociation constant (Kd) of 3μM. This allows the cytochrome to be exported in an unfolded state and heme to be added exogenously to initiate correct folding of the cytochrome by burying hydrophobic side chains (Robinson et al., 1997). Furthermore, heme binding to cytochrome <i>b</i><sub>562</sub> has a high affinity interaction with a dissociation constant (Kd) of 9nM at 25°C (Robinson et al., 1997). This BioBrick part has been inserted into the pSB1C3 backbone and uses the promoter [https://parts.igem.org/Part:BBa_J23104 BBa_J23104]. This part can be used in the VS45 strain of E.coli containing deletions that prevent amyloid from binding to the outside of the cell and increase the rate of protein exiting the cell via the curli export system.  
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We chose Cytochrome <i>b</i><sub>562</sub> as the electron carrier to make our amyloid conductive. The structure of cytochrome <i>b</i><sub>562</sub> consists of a single 24kDa subunit containing four nearly parallel alpha helices (Fujiwara, Fnkumori, and Yamanaka, 1993; Mathews et al., 1979). B-type cytochromes are a favourable choice because haem binds in a non-ionic fashion to the two ligands Methionine-7 and Histidine-106 (Xavier et al., 1978). Haem binding has been shown to occur in both the native protein and the denatured protein, although the latter exhibits a modest affinity with a dissociation constant (Kd) of 3μM. This allows the cytochrome to be exported in an unfolded state and haem to be added exogenously to initiate correct folding of the cytochrome by burying hydrophobic side chains (Robinson et al., 1997). Furthermore, haem binding to cytochrome <i>b</i><sub>562</sub> has a high affinity interaction with a dissociation constant (Kd) of 9nM at 25°C (Robinson et al., 1997). This BioBrick has been optimised for use in the VS45 strain of E.coli containing deletions that prevent amyloid from binding to the outside of the cell and increase the rate of protein exiting the cell via the curli export system.
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===Validation===
 
===Validation===
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Validation of our fusion protein’s export was achieved using four techniques. We first analysed it via gel electrophoresis (fig.2). By comparing the sizes of the fragments to a marker we discovered the size of our fragment ( as seen in figure x) to be around 1400 base pairs, matching the actual size of 1339 base pairs of the CsgA<sub>ss</sub>, Sup35NM and Cytochrome <i>b</i><sub>562</sub> sequences. Also, our gel shows a fragment to be about 2000 base pairs, confirming it is PSB1c3 which is 2030 base pairs in size.
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Validation of our fusion protein’s export was achieved using four techniques.  
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===Plasmid===
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We first analysed the biobrick by diagnostic restriction digest followed by gel electrophoresis (Fig. 1). By comparing the sizes of the fragments to a marker we validated the size of our fragment (as seen in figure 1) to be consistent with size of 1339 base pairs of the CsgAss, Sup35NM and Cytochrome <i>b</i><sub>562</sub> sequences.
  
 
[[File:Team Kent Envirowire gel2.jpg|thumb|center|400px|Figure 1. Agarose gel of the restriction digest of BBa_K1739003 in pSCB13 plasmid backbone with EcoRI and PstI.]]
 
[[File:Team Kent Envirowire gel2.jpg|thumb|center|400px|Figure 1. Agarose gel of the restriction digest of BBa_K1739003 in pSCB13 plasmid backbone with EcoRI and PstI.]]
  
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We then validated our part using a segmented diagnostic Congo Red agar plate with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains transformed with our plasmid. As a negative control, we used VS45 with pVS105, as this plasmid contains CsgA<sub>ss</sub> and Sup35M, it does not have the ability to export amyloid-forming proteins. These strains were plated in 2 quarter segments of the plate and left to incubate for 24 hours at 37°C. This resulted red colonies of VS45 with CsgA<sub>ss</sub>-Sup35NM-Cyt<i>b562</i> due to the binding of Congo Red to the amyloid fibres produced by the colonies. White colonies of VS45 with the negative control plasmid, as expected, showed no export of amyloid-forming protein (shown in fig.1). These results confirmed that our protein was being produced and targeted to the curli export pathway by csgA, as well as self-assembling into amyloid fibres.
 
  
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[[File:Team Kent platescomparison.jpg|thumb|center|400px|Figure 2. Shows our Congo red plates. The top plate depicts both our negative control plasmid, PVS105 and our BioBrick containing CsgAss and Sup35NM. Whereas the bottom plate shows only VS45 with our BioBrick. The presence of red colonies illustrates the successful formation of amyloid.]]
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===Congo Red plate assay===
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We then validated our part using a Congo Red agar plate assay with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains expression our fusion protein. As a negative control, we used VS45 with pVS105, as this plasmid contains CsgAss and Sup35M, it does not have the ability to self-assemble into amyloid nano-wires. These strains were plated in 2 quarter segments of the plate and left to incubate for 24 hours at 37°C. This resulted red colonies of VS45 with CsgAss-Sup35NM-Cyt <i>b</i><sub>562</sub> due to the binding of Congo Red to the amyloid fibres produced by the colonies. White colonies of VS45 with the negative control plasmid, as expected, showed no export of amyloid-forming protein (shown in fig.1). These results confirmed that our fusion protein was being produced and targeted to the curli export pathway by CsgA<sub>ss</sub>, as well as self-assembling into amyloid fibres.
  
  
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The third method of validation for protein export and amyloid formation was achieved using atomic force microscopy (AFM) imaging to provide a topography of our samples. Using the aforementioned E.coli strains a, 5 day incubation at 25°C was carried out to make sure that the amyloid fibres were stable for the AFM protocol, as suggested by Sivanathan and Hochschild (2012). The resulting images clearly showed the presence of amyloid fibres in the VS45 sample with PVS72 (figures 3 and 4) and no amyloid fibres in the VS45 with the negative control plasmid (figure 5).
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[[File:Team Kent platescomparison.jpg|thumb|center|400px|Figure 2. Shows our Congo red plates. The top plate depicts both our negative control plasmid, PVS105 and our BioBrick containing CsgAss and Sup35NM. Whereas the bottom plate shows only VS45 with our BioBrick. The presence of red colonies illustrates the successful formation of amyloid.]]
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===AFM Imaging===
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Further validation for protein export and amyloid formation by our fusion protein was achieved using atomic force microscopy (AFM) imaging of our samples. Using the aforementioned E.coli strains a 5 day incubation at 25°C was carried out to make sure that the amyloid fibres were stable for the AFM protocol, as suggested by Sivanathan and Hochschild (2012). The resulting images showed the presence of amyloid aggregates in the VS45 sample with PVS72 (figures 3 and 4) compared with samples without amyloid fibres in the VS45 with the negative control plasmid (figure 5), although the nano-wire were assembled less efficiently resulting in less straight, shorter and more clumped fibrils than with Sup35NM alone.
  
 
[[File:Team KentAFMwithcyt.jpg|thumb|center|400px|Figure 3. Shows AFM imaging of our Envirowire plasmid. The nano-wires are visible although the reduced assembly efficiency of our designed amyloid nano-wire may be due to the incomplete folding of cytochrome <i>b</i><sub>562</sub> as the heme needed for complete folding wasn’t present in the growth media.]]
 
[[File:Team KentAFMwithcyt.jpg|thumb|center|400px|Figure 3. Shows AFM imaging of our Envirowire plasmid. The nano-wires are visible although the reduced assembly efficiency of our designed amyloid nano-wire may be due to the incomplete folding of cytochrome <i>b</i><sub>562</sub> as the heme needed for complete folding wasn’t present in the growth media.]]
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Further validation was required to ensure that our cytochrome was attached to the Sup35-NM monomers and had bound to the haem. This was achieved by carrying out a conductivity test. Firstly a biofilm formation was induced on a piece of acrylic before the conductance was measured over the length of the biofilm (1 inch), the results, however, were inconclusive. Future testing could be done to assess the affect of biofilm length and diameter on conductance and resistance to optimise electron transmission efficiency.  
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===Heme plate and conductivity assay===
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Further validation was performed to ensure that our cytochrome attached to the Sup35NM nano-wire had bound to the heme and conferred conductive properties. This was achieved by carrying out a conductivity test. Firstly a biofilm formation was induced on a agar plate before the conductance was measured over the length of the biofilm (1 inch), the results, however, were inconclusive. Future testing on individual nano-wire could be done to assess the affect of biofilm length and diameter on conductance and resistance to optimize assembly and conductivity efficiency.
  
  

Revision as of 21:09, 18 September 2015

Sequence coding for Envirowire: Sup35NM with N-terminal CsgAss and C-terminal cytb562

This BioBrick encodes for the Envirowire fusion protein. It contains three segments, CsgA signal sequence, Sup35NM (https://parts.igem.org/wiki/index.php?title=Part:BBa_K1739000 BBa_K1739000] and https://parts.igem.org/wiki/index.php?title=Part:BBa_K1739002 BBa_K1739002]) and Cytochrome b562 (BBa_K1739001) The bipartite CsgA signal sequence targets the Sec protein export pathway followed by the endogenous curli export system of E.coli allowing our protein to be easily exported into an external medium (Sivanathan and Hochschild, 2012; Sivanathan and Hochschild, 2013). Sup35NM is derived from the yeast prion protein Sup35p and excludes the C-terminal domain. The N-terminal domain allows self-assembly of functional amyloid (Frederick et al., 2014; Glover et al. 1997). This has previously been discussed by Tessier and Lindquist (2009) who show that two beta-sheets bond together in a self-complimenting ‘steric zipper’ that excludes water, leaving a highly stable parallel beta-sheet with one molecule every 4.7Å. The particular advantage of using Sup35-NM is that in its native state Sup35p has two functional domains, the N and C terminal, separated by the highly charged M domain (Frederick et al., 2014; Glover et al. 1997; Wickner et al., 2007). ). Here, with the removal of the functional C domain allows us to add our own functional protein, in this case cytochrome b562, to form a fusion protein. We chose Cytochrome b562 as the electron carrier to make our amyloid conductive. The structure of cytochrome b562 consists of a single 24kDa subunit containing four nearly parallel alpha helices (Fujiwara, Fnkumori, and Yamanaka, 1993; Mathews et al., 1979). B-type cytochromes are a favourable choice because heme binds in a non-ionic fashion to the two ligands Methionine-7 and Histidine-106 (Xavier et al., 1978). Heme binding has been shown to occur in both the native protein and the denatured protein, although the latter exhibits a modest affinity with a dissociation constant (Kd) of 3μM. This allows the cytochrome to be exported in an unfolded state and heme to be added exogenously to initiate correct folding of the cytochrome by burying hydrophobic side chains (Robinson et al., 1997). Furthermore, heme binding to cytochrome b562 has a high affinity interaction with a dissociation constant (Kd) of 9nM at 25°C (Robinson et al., 1997). This BioBrick part has been inserted into the pSB1C3 backbone and uses the promoter BBa_J23104. This part can be used in the VS45 strain of E.coli containing deletions that prevent amyloid from binding to the outside of the cell and increase the rate of protein exiting the cell via the curli export system.


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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 718


Validation

Validation of our fusion protein’s export was achieved using four techniques.


Plasmid

We first analysed the biobrick by diagnostic restriction digest followed by gel electrophoresis (Fig. 1). By comparing the sizes of the fragments to a marker we validated the size of our fragment (as seen in figure 1) to be consistent with size of 1339 base pairs of the CsgAss, Sup35NM and Cytochrome b562 sequences.

Figure 1. Agarose gel of the restriction digest of BBa_K1739003 in pSCB13 plasmid backbone with EcoRI and PstI.


Congo Red plate assay

We then validated our part using a Congo Red agar plate assay with the antibiotics chloramphenicol and ampicillin present in order to select the VS45 strains expression our fusion protein. As a negative control, we used VS45 with pVS105, as this plasmid contains CsgAss and Sup35M, it does not have the ability to self-assemble into amyloid nano-wires. These strains were plated in 2 quarter segments of the plate and left to incubate for 24 hours at 37°C. This resulted red colonies of VS45 with CsgAss-Sup35NM-Cyt b562 due to the binding of Congo Red to the amyloid fibres produced by the colonies. White colonies of VS45 with the negative control plasmid, as expected, showed no export of amyloid-forming protein (shown in fig.1). These results confirmed that our fusion protein was being produced and targeted to the curli export pathway by CsgAss, as well as self-assembling into amyloid fibres.


Figure 2. Shows our Congo red plates. The top plate depicts both our negative control plasmid, PVS105 and our BioBrick containing CsgAss and Sup35NM. Whereas the bottom plate shows only VS45 with our BioBrick. The presence of red colonies illustrates the successful formation of amyloid.

AFM Imaging

Further validation for protein export and amyloid formation by our fusion protein was achieved using atomic force microscopy (AFM) imaging of our samples. Using the aforementioned E.coli strains a 5 day incubation at 25°C was carried out to make sure that the amyloid fibres were stable for the AFM protocol, as suggested by Sivanathan and Hochschild (2012). The resulting images showed the presence of amyloid aggregates in the VS45 sample with PVS72 (figures 3 and 4) compared with samples without amyloid fibres in the VS45 with the negative control plasmid (figure 5), although the nano-wire were assembled less efficiently resulting in less straight, shorter and more clumped fibrils than with Sup35NM alone.

Figure 3. Shows AFM imaging of our Envirowire plasmid. The nano-wires are visible although the reduced assembly efficiency of our designed amyloid nano-wire may be due to the incomplete folding of cytochrome b562 as the heme needed for complete folding wasn’t present in the growth media.
Figure 4. Illustrates a cluster of amyloid fibers, it is zoomed in from figure 3.
Figure 5. Shows AFM imaging of our negative control. No nano-wires are present.


Heme plate and conductivity assay

Further validation was performed to ensure that our cytochrome attached to the Sup35NM nano-wire had bound to the heme and conferred conductive properties. This was achieved by carrying out a conductivity test. Firstly a biofilm formation was induced on a agar plate before the conductance was measured over the length of the biofilm (1 inch), the results, however, were inconclusive. Future testing on individual nano-wire could be done to assess the affect of biofilm length and diameter on conductance and resistance to optimize assembly and conductivity efficiency.


References

Frederick, K., Debelouchina, G., Kayatekin, C., Dorminy, T., Jacavone, A., Griffin, R. and Lindquist, S. (2014). Distinct Prion Strains Are Defined by Amyloid Core Structure and Chaperone Binding Site Dynamics. Chemistry & Biology, 21(2), pp.295-305.

Fujiwara, T., Fnkumori,, Y. and Yamanaka, T. (1993). Halobacterium halobium Cytochrome b-558 and Cytochrome b-562: Purification and Some Properties. J. Biochem., 113, pp.48-54.

Glover, J., Kowal, A., Schirmer, E., Patino, M., Liu, J. and Lindquist, S. (1997). Self-Seeded Fibers Formed by Sup35, the Protein Determinant of [PSI+], a Heritable Prion-like Factor of S. cerevisiae. Cell, 89(5), pp.811-819.

Mathews, F. S., Bethge, P. H., & Czerwinski, E. W. (1979). The structure of cytochrome b562 from Escherichia coli at 2.5 A resolution. Journal of Biological Chemistry, 254(5), 1699-1706.

Robinson, C., Liu, Y., Thomson, J., Sturtevant, J. and Sligar, S. (1997). Energetics of Heme Binding to Native and Denatured States of Cytochrome b 562 †. Biochemistry, 36(51), pp.16141-16146.

Sivanathan, V. and Hochschild, A. (2012). Generating extracellular amyloid aggregates using E. coli cells. Genes & Development, 26(23), pp.2659-2667.

Sivanathan, V. and Hochschild, A. (2013). A bacterial export system for generating extracellular amyloid aggregates. Nat Protoc, 8(7), pp.1381-1390.

Tessier, P. and Lindquist, S. (2009). Unraveling infectious structures, strain variants and species barriers for the yeast prion [PSI+]. Nat Struct Mol Biol, 16(6), pp.598-605.

Wickner, R., Edskes, H., Shewmaker, F. and Nakayashiki, T. (2007). Prions of fungi: inherited structures and biological roles. Nature Reviews Microbiology, 5(8), pp.611-618.

Xavier, A., Czerwinski, E., Bethge, P. and Mathews, F. (1978). Identification of the haem ligands of cytochrome b562 by X-ray and NMR methods. Nature, 275(5677), pp.245-247.