Part:BBa_K538000

Cpn10 (O. antarctica)

Figure 1: (a) E. coli 's growth rate as a function of temperature, in strains that do (white circles) or do not (black circles) express Cpn60/10, and (b) in vitro refolding activities of Cpn60/10 as a function of temperature (black circles), compared with that of E. coli 's endogenous GroEL/ES (white circles). Adapted from Ferrer et al. (2003)

This BioBrick encodes the protein cochaperonin 10, which is part of the Cpn60/10 chaperone system of Oleispira antarctica. When coexpressed with chaperonin 60 (Cpn60, BBa_K538001), it forms a heteromultimer that can refold enzymes at very low temperatures, thus safeguarding their functionality. This has been shown to enable E. coli to grow even at freezing point. (Figure 1)

Usage

This part is used in several cold resistance BioBricks, or CryoBricks, developed by team [http://2011.igem.org/Team:Amsterdam Amsterdam 2011]. At the time of this writing, these parts contain both BBa_K538000 and BBa_K538001:

• BBa_K538210: a Cpn10/60 operon with the pLacI promoter
• BBa_K538211: a Cpn60/10 operon with the pLacI promoter
• BBa_K538310: a Cpn10/60 operon with the pBAD promoter
• BBa_K538311: a Cpn60/10 operon with the pBAD promoter

For an up-to-date list of all parts containing just BBa_K538000, refer to the related parts page.

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

1. Gething & Sambrook Protein folding in the cell, Nature 355, 33–45 (1992)
2. Walter & Buchner Molecular Chaperones — Cellular Machines for Protein Folding, Angew. Chem. Int. Ed. Eng. 41, 1098–1113 (2002)
3. Ferrer et al. Chaperonins govern growth of Escherichia coli at low temperatures, Nat. Biotech. 21, 1266 - 1267 (2003)
4. 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)

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 52
  

Functional Parameters: Austin_UTexas

BBa_K538000 parameters

Burden Imposed by this Part:

Burden Value: -0.2 ± 4%

Burden is the percent reduction in the growth rate of E. coli cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the BBa_K3174002 - BBa_K3174007 pages for more information on the methods, an explanation of the sources of burden, and other conclusions from a large-scale measurement project conducted by the 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.