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Basic Parts

Three different types of basic parts were used to construct our CryoBricks: promoters, ribosome binding sites (RBSes) and coding regions. This page contains information on their purpose in the project. Emphasis is placed on the reason why we selected these particular parts, and in case of the coding regions, the biological function of the encoded protein. For more information on the constructs into which these parts were combined, refer to the composite parts page.

Promoters

Several criteria were considered in selecting promoters for use in our CryoBricks. An ideal promoter would be: available from the parts registry, so that we can easily obtain and incorporate it; active at low temperatures, so that it works when we need it to; and positively regulated, so it won't be active unless we want it to. A search for promoters that matched one or more of these criteria resulted in the following selection:

pLacI

The Lac operon's promoter, which is often used to induce expression of proteins from plasmids artificially transformed into E. coli. Literature has numerous examples of this promoter being used to (over)express proteins at low temperatures, and it's available from the registry as [http://partsregistry.org/Part:BBa_R0010 BBa_R0010]. Unfortunately, it's not positively regulated, but its activity can be controlled to a certain extent. It's negatively regulated by the LacI protein, and as such can be stimulated by repressing LacI with IPTG or lactose. However, it will be active constitutively in strains that don't express LacI.

pBAD

The promoter of the araBAD operon. Its biological function is similar to that of pLacI; in its natural context, it activates transcription of enzymes involved with the consumption of a carbon source, when this carbon source is available to the cell. Its activity is mediated by the AraC protein, which either activates of represses expression, depending on the concentration of L-arabinose.[http://www.pnas.org/content/85/15/5444.full.pdf] The pBAD promoter is available from the registry as [http://partsregistry.org/Part:BBa_I13453 BBa_I13453], but team British Columbia 2009 derived two new promoters from this part, responding either more or less strongly to the presence of arabinose (Stronger response: [http://partsregistry.org/Part:BBa_K206000 BBa_K206000], weaker response: [http://partsregistry.org/Part:BBa_K206001 BBa_K206001]). These pBAD derived promoters were selected because of their different arabinose sensitivity. This difference can facilitate finetuning the expression levels of proteins while using a single inducer, which can be further tweaked by combining the promoters with different RBSes.

hybB

The promoter of the hydrogenase II operon. It's also known as the coldshock promoter, because it's inactive at 37°C and activates at temperatures below 30°C. Using this promoter would allow us to only have our cells express CryoBricks when E. coli actually benefits from this, but makes inducing expression in a dose-dependent manner impossible, as it's not induced by a molecule we add. It enables CryoBrick expression to be directed on 'autopilot', which has pros (for example, in large-scale industrial applications) and cons (in case CryoBricks comprising such a promoter cross over to an uncontrolled environment, be it by accident or design). The registry submission of this brick, [http://partsregistry.org/Part:BBa_J45503 BBa_J45503], never made it past the planning stage, but the brick is incorporated in several composite parts.

Final selection

All selected promoters fulfilled the availability criterion, being submitted to the registry, but hybB was only available as part of an assembled construct. The construct was ordered and preparations were made to PCR it out of this construct, but constraints on time and safety considerations caused us to decide against using it in the end.

The project was supposed to include a characterisation experiment to quantify the remaining promoters' strengths at low temperatures. Unfortunately, technical difficulties with cloning, and subsequent assembly of the reporter constructs required for this, made such characterisation infeasible. Even though we couldn't confirm pLacI and pBAD's activity at low temperatures, it was decided to construct our CryoBricks with both promoters. Of the available pBAD promoters, we selected the one that responded most strongly to arabinose ([http://partsregistry.org/Part:BBa_K206000 BBa_K206000]). It allows for the highest the maximum expression level, and any of the expression levels attainable with the related promoters can be achieved as well, given a finely tuned concentration of arabinose.

RBSes

Similar to our promoter selection criteria, the ideal RBS for our CryoBricks should be active at low temperatures, and available from the registry. We suspect temperature may have quite a drastic impact on how an RBS functions; RBSes form structures in mRNA that mediate ribosome binding, but thermodynamics govern the dynamicity (and stability) of such structures. Literature study revealed only very little information about how temperature affects the activity of RBSes. To fill this gap in the knowledgebase, we planned to characterise some of the RBSes available from the registry, using reporter constructs to measure their strength at low temperatures. Unfortunately, technical difficulties and tight deadlines impaired these plans as they did with the plans to characterise promoter strengths.

Forced to omit characterising the strength of different RBSes at low temperatures, we decided to take our chances with the RBS team Warsaw 2010 defined as their standard against which to compare other RBS strengths ([http://partsregistry.org/Part:BBa_B0034 BBa_B0034]). Running on the crude assumption low temperatures affect all RBSes in an equivalent manner, we reasoned this RBS - which is relatively strong compared to other RBSes at 'normal' temperatures - should be relatively strong at low temperatures too.

Coding regions

The novelty of the icE. coli project lies in its submissions to the registry: 5 different coding regions, each encoding a protein reported to enhance cold resistance one way or another. Cold resistance, in this context, is defined as protein mediated mitigation of cold-induced systems failure. Such systems failure can be caused on many different levels, such as a decrease in the fluidity of the membrane; an excessive increase in structural rigidity of proteins, DNA and RNA; a drop in the rate of (bio)chemical reactions; and in extreme cases, mechanical damage caused by ice crystallisation. More often than not, low temperatures cause failure on several levels simultaneously, and mitigation of the effect on one level may be cancelled out by failure of another. In some cases, though, systems failure may be limited or prevented entirely by the expression of specific (combinations of) proteins.

Literature study reveals several proteins have been reported to decrease E. coli 's sensitivity to low temperatures. Team Amsterdam selected 5 of these proteins to create cold resistance BioBricks, or CryoBricks. They are all chaperones, originally found in cryophillic bacteria which thrive in cold environments, like the Arctic. The selected proteins are:

• Cpn10 and Cpn60, from Oleispira antarctica
• SheDnaK, from Shewanella sp. AC10
• CspA and CspC, from Polaribacter irgensii

All these proteins are, at least to a certain degree, homologous to chaperone proteins endogenous to E. coli. In addition, function loss of these endogenous chaperones is associated with cold-induced systems failure. Theoretically, expression of the cryophile chaperones listed above compensates for the loss of function endogenous chaperones experience at low temperatures, in doing so mitigating the impact of these temperatures on cellular activity.

Note that because of the many different levels at which cold-induced systems failure can occur, many different ways of characterising cold resistance are employed in different articles. Two prevalent ones used to characterise our CryoBricks are:

• the effect expression of a (combination of) protein(s) has on an organism's ability to survive freezing and thawing
• the effect expression of a (combination of) protein(s) has on an organism's specific growth rate at suboptimal temperatures

Below, you'll find information on the biology of the selected chaperones; how they contribute to cold resistance in E. coli. For more information on the methods and results of characterising them, refer to the CryoBrick characterisation page.

Cpn10 and Cpn60

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.

References

SheDnaK

CspA and CspC

References

1. Huo, Martin & Schleif Alternative DNA loops regulate the arabinose operon in Escherichia coli, Procl. Natl. Acad. Sci. USA 85, 5444-5448 (1988)
2. Gething & Sambrook Protein folding in the cell, Nature 355, 33–45 (1992)
3. Walter & Buchner Molecular Chaperones — Cellular Machines for Protein Folding, Angew. Chem. Int. Ed. Eng. 41, 1098–1113 (2002)
4. Ferrer et al. Chaperonins govern growth of Escherichia coli at low temperatures, Nat. Biotech. 21, 1266 - 1267 (2003)
5. 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)

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