Difference between revisions of "Template:2011/Team:Amsterdam/Cpn60 10"

Latest revision as of 07:41, 21 September 2011

Biology

Cpn10 and Cpn60 are homologous to GroES and GroEL of E. coli, respectively. The GroEL/ES chaperone system promotes the folding and/or assembly of over 30% of E. coli's cellular proteins, is required for bacteriophage morphogenesis and has a role in protein secretion.[http://www.nature.com/nature/journal/v355/n6355/abs/355033a0.html][http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820020402%2941:7%3C1098::AID-ANIE1098%3E3.0.CO;2-9/abstract] However, it rapidly loses its refolding activity at temperatures below 37°C.[http://www.nature.com/nbt/journal/v21/n11/pdf/nbt1103-1266b.pdf] (Figure 1b) Cpn60/10, on the other hand, functions very well at these temperatures.

Ferrer et al. identified 22 housekeeping proteins involved with E. coli 's systems failure at low temperatures. They went on to demonstrate their interactions with chaperones are key determinants of activity at these temperatures. It was shown that the Cpn60 protein from O. antarctica coprecipitates with many of the proteins found in the proteome of E. coli.[http://onlinelibrary.wiley.com/doi/10.1002/pmic.200500031/abstract] Also, by transforming E. coli with Cpn10 and Cpn60, they've enabled it to grow even at freezing point. (Figure 1a) Their findings suggest inactivation of a few cold-sensitive key proteins causes systems failure in E. coli, and that cells may be 'rescued' by reactivating these genes.

Characterisation

Ferrer et al. have already shown the Cpn60/10 system to have a nameworthy impact on E. coli's cold tolerance. Unfortunately, this brick's developers only managed to assemble a protein generator comprising Cpn10 's coding region. Reproducing the results of Ferrer et al. would have required coexpression of Cpn10 and Cpn60, so this impossible.

Attempts were made to characterise whether or not Cpn10 expression can bestow a certain degree of cold resistance independent of Cpn60. According to expectation, Cpn10 doesn't significantly affect E. coli 's specific growth rate at suboptimal temperatures. Refer to the part's experience page for the graph summarizing the Cpn10 generator's growth rate investigation.

In addition to investigating the effect of Cpn10 on growth rate a suboptimal temperatures, the effect its expression has on freeze/thaw cycle survival was characterised. During this experiment, a most remarkable observation was made; the data suggests Cpn10 enhances E. coli 's freeze/thaw survival rate in and of itself, without being coexpressed with Cpn60. Experiments to verify or falsify this suggestion are currently being prepared and executed. This section will be updated in due time.

Safety

There are risks involved with enhancing bacterial cold resistance and facilitating their growth at low temperatures. Please refer to the [http://2011.igem.org/Team:Amsterdam/Project/Safety safety page] of this brick's developers.

References

Gething & Sambrook Protein folding in the cell, Nature 355, 33–45 (1992)
Walter & Buchner Molecular Chaperones — Cellular Machines for Protein Folding, Angew. Chem. Int. Ed. Eng. 41, 1098–1113 (2002)
Ferrer et al. Chaperonins govern growth of Escherichia coli at low temperatures, Nat. Biotech. 21, 1266 - 1267 (2003)
Strocchi, Ferrer, Timmis & Golyshin Low temperature-induced systems failure in Escherichia coli: Insights from rescue by cold-adapted chaperones, Proteomics 6 (1), 193-206 (2005)

@@ Line 3: / Line 3: @@
 ''Cpn10'' and ''Cpn60'' are homologous to ''GroES'' and ''GroEL'' of ''E. coli'', respectively. The ''GroEL/ES'' chaperone system promotes the folding and/or assembly of over 30% of ''E. coli'''s cellular proteins, is required for bacteriophage morphogenesis and has a role in protein secretion.[http://www.nature.com/nature/journal/v355/n6355/abs/355033a0.html][http://onlinelibrary.wiley.com/doi/10.1002/1521-3773%2820020402%2941:7%3C1098::AID-ANIE1098%3E3.0.CO;2-9/abstract] However, it rapidly loses its refolding activity at temperatures below 37°C.[http://www.nature.com/nbt/journal/v21/n11/pdf/nbt1103-1266b.pdf] (Figure 1b) ''Cpn60/10'', on the other hand, functions very well at these temperatures.
-Ferrer ''et al.'' identified 22 housekeeping proteins involved with ''E. coli'' 's systems failure at low temperatures. They went on to demonstrate their interactions with chaperones are key determinants of activity at these temperatures. It was shown that the ''Cpn60'' protein from ''O. antarctica'' coprecipitates with many of the proteins found in the proteome of ''E. coli''.[http://onlinelibrary.wiley.com/doi/10.1002/pmic.200500031/abstract] Also, by transforming ''E. coli'' with ''Cpn10'' and ''Cpn60'', they've enabled it to grow even at freezing point. (Figure 1a) Their findings suggest inactivation of a few cold-sensitive key causes systems failure in ''E. coli'', and that cells may be 'rescued' by reactivating these genes.
+Ferrer ''et al.'' identified 22 housekeeping proteins involved with ''E. coli'' 's systems failure at low temperatures. They went on to demonstrate their interactions with chaperones are key determinants of activity at these temperatures. It was shown that the ''Cpn60'' protein from ''O. antarctica'' coprecipitates with many of the proteins found in the proteome of ''E. coli''.[http://onlinelibrary.wiley.com/doi/10.1002/pmic.200500031/abstract] Also, by transforming ''E. coli'' with ''Cpn10'' and ''Cpn60'', they've enabled it to grow even at freezing point. (Figure 1a) Their findings suggest inactivation of a few cold-sensitive key proteins causes systems failure in ''E. coli'', and that cells may be 'rescued' by reactivating these genes.
 ===Characterisation===
-Ferrer ''et al.'' have already shown the ''Cpn60/10'' system to have a nameworthy impact on ''E. coli'''s cold tolerance. It is currently being investigated whether or not the CryoBricks comprising [[Part:BBa_K538000 | BBa_K538000]] and [[Part:BBa_K538001 | BBa_K538001]] reproduce this effect.
+Ferrer ''et al.'' have already shown the ''Cpn60/10'' system to have a nameworthy impact on ''E. coli'''s cold tolerance. Unfortunately, this brick's developers only managed to assemble a protein generator comprising ''Cpn10'' 's coding region. Reproducing the results of Ferrer ''et al.'' would have required coexpression of ''Cpn10'' and ''Cpn60'', so this impossible.
+Attempts '''were''' made to characterise whether or not ''Cpn10'' expression can bestow a certain degree of cold resistance independent of ''Cpn60''. According to expectation, ''Cpn10'' doesn't significantly affect ''E. coli'' 's specific growth rate at suboptimal temperatures. Refer to the part's [[Part:BBa_K538000:Experience|experience]] page for the graph summarizing the ''Cpn10'' generator's growth rate investigation.
-'''[Update pending; waiting for lab results]'''
+In addition to investigating the effect of ''Cpn10'' on growth rate a suboptimal temperatures, the effect its expression has on freeze/thaw cycle survival was characterised. During this experiment, a most remarkable observation was made; the data suggests ''Cpn10'' enhances ''E. coli'' 's  freeze/thaw survival rate in and of itself, without being coexpressed with ''Cpn60''. Experiments to verify or falsify this suggestion are currently being prepared and executed. This section will be updated in due time.
-In case the chaperones' effect on growth rate at low temperatures '''is''' reproducible, team [http://2011.igem.org/Team:Amsterdam Amsterdam] expects a similar result when transforming related species with their CryoBricks. If systems failure is caused by inactivation of genes like ''Dps'', ''ClpB'', ''DnaK'' and ''RpsB'' (or their homologs), ''Cpn60/10'' might be able to recover their activity. This brick's developers had intended to investigate this in some bacterial species, but constraints on time made this unfeasible.