Part:BBa_K538002

SheDnaK (Shewanella sp. Ac10)

Figure 1: ATPase activity as a function of temperature, for the DnaK of Shewanella sp. Ac10 (SheDnaK, white squares) and E. coli (EcoDnaK, black circles). Adapted from Yoshimune et al. (2005)

This BioBrick encodes the DnaK protein of Shewanella sp. Ac10. It's homologous to E. coli's endogenous DnaK (EcoDnaK), which plays a role in many different cellular processes. Refer to the Biology section below for a few examples of this. Shewanella sp. Ac10 DnaK, better known as SheDnaK, was used by Yoshimune et al. to rescue an E. coli DnaK-null mutant, allowing it to grow at 15°C.

Usage

Team [http://2011.igem.org/Team:Amsterdam Amsterdam 2011] developed two protein generators incorporating BBa_K538002. These CryoBricks - cold resistance enhancing BioBricks - both contain a promoter, an RBS and this brick's SheDnaK coding region. In BBa_K538202, this part is placed under control of the Lac operon's promoter. BBa_K538302 contains it under control of team [http://2009.igem.org/Team:British_Columbia British Columbia 2009]'s pBAD derivative (arabinose inducible) instead.

Biology

DnaK is a 70 kilodalton heatshock protein involved with many different processes. It's E. coli's primary Hsp70 homolog[http://www.jbc.org/content/270/18/10412.full], and its function is mostly regulated by interactions with the smaller heatshock proteins DnaJ and GrpE. It's been debated whether or not DnaK is involved with assembling the ribosome's 30S subunit, but Maki, Southworth and Culver conclusively demonstrated this to be the case.[http://rnajournal.cshlp.org/content/9/12/1418.full] It is also essential for lambda phage propagation, playing a key role in its replication by binding the lambda P protein, disassembling the lambda P:DnaB complex.[http://www.jbc.org/content/268/7/4821.abstract]

Cold-induced inactivation of EcoDnaK, combined with the inability to refold it, is reported to be a causative agent of systems failure in E. coli at low temperatures.[http://onlinelibrary.wiley.com/doi/10.1002/pmic.200500031/abstract] Yoshimune et al. isolated SheDnaK from the Antarctic Shewanella bacterium, and observed it has a much higher ATPase activity at low temperatures than EcoDnaK, which is characteristic for cold-active enzymes.[http://www.springerlink.com/content/f50n0gahppxacytb/] (Figure 1) They subsequently expressed SheDnaK in a DnaK-null mutant. This did not rescue E. coli 's growth at 43°C, nor did the strain support lambda phage propagation at 30°C, but it did allow their SheDnaK comprising E. coli to grow at temperatures as low as 15°C, which was impossible for the null-mutant.

Team [http://2011.igem.org/Team:Amsterdam Amsterdam 2011] tried to enhance E. coli 's growth rate at low temperatures by expressing SheDnaK, speculating it might take over the various functions lost at these temperatures by EcoDnaK's unfolding.

Characterisation

[Update pending]

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. Ziegelhoffer, Lopez-Buesa & Craig The Dissociation of ATP from hsp70 of Saccharomyces cerevisiae Is Stimulated by Both Ydj1p and Peptide Substrates, J. of Bio. Chem 270, 10412-10419 (1995)
2. Maki, Southworth & Culver Demonstration of the role of the DnaK chaperone system in assembly of 30S ribosomal subunits using a purified in vitro system, RNA 9, 1418-1421 (2003)
3. Osipiuk, Georgopoulos & Zylicz Initiation of lambda DNA replication - The Escherichia coli small heat shock proteins, DnaJ and GrpE, increase DnaK's affinity for the lambda P protein, J. of Bio. Chem 268, 4821-4827 (1993)
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)
5. Yoshimune et al. Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures Extremophiles 9 (2), 145-150 (2005)

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 747
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]